Vitamin receptor drug delivery conjugates for treating inflammation

ABSTRACT

Described herein are compositions, methods, compounds, conjugates, and kits for use in targeted drug delivery using drug delivery conjugates containing hydrophilic spacer linkers for use in treating disease states caused by pathogenic cell populations, such as inflammatory cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in part of U.S. application Ser. No.13/518,291, filed Jun. 21, 2012, which is a U.S. national applicationunder 35 U.S.C. § 371(b) of International Application Serial No.PCT/US2010/061897 filed Dec. 22, 2010, and claims priority under 35 USC§ 119(e) to U.S. Provisional Application Ser. No. 61/289,952, filed onDec. 23, 2009, U.S. Provisional Application Ser. No. 61/291,103 filed onDec. 30, 2009, U.S. Provisional Application Ser. No. 61/351,032, filedon Jun. 3, 2010, U.S. Provisional Application Ser. No. 61/374,830, filedon Aug. 18, 2010, U.S. Provisional Application Ser. No. 61/386,785,filed on Sep. 27, 2010, and U.S. Provisional Application Ser. No.61/391,230, filed on Oct. 8, 2010, the entire disclosures of each ofwhich are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to compositions and methods for use intargeted drug delivery. More particularly, the invention is directed tocell-surface receptor binding drug delivery conjugates containinghydrophilic spacer linkers for use in treating disease states caused bypathogenic cell populations and to methods and pharmaceuticalcompositions that use and include such conjugates.

BACKGROUND

The mammalian immune system provides a means for the recognition andelimination of foreign pathogens. While the immune system normallyprovides a line of defense against foreign pathogens, there are manyinstances where the immune response itself is involved in theprogression of disease. Exemplary of diseases caused or worsened by thehost's own immune response are autoimmune diseases and other diseases inwhich the immune response contributes to pathogenesis. For example,macrophages are generally the first cells to encounter foreignpathogens, and accordingly, they play an important role in the immuneresponse, but activated macrophages can also contribute to thepathophysiology of disease in some instances.

The folate receptor is a 38 KD GPI-anchored protein that binds thevitamin folic acid with high affinity (<1 nM). Following receptorbinding, rapid endocytosis delivers the vitamin into the cell, where itis unloaded in an endosomal compartment at low pH. Importantly, covalentconjugation of small molecules, proteins, and even liposomes to folicacid does not block the vitamin's ability to bind the folate receptor,and therefore, folate-drug conjugates can readily be delivered to andcan enter cells by receptor-mediated endocytosis.

Because most cells use an unrelated reduced folate carrier to acquirethe necessary folic acid, expression of the folate receptor isrestricted to a few cell types. With the exception of kidney, choroidplexus, and placenta, normal tissues express low or nondetectable levelsof the folate receptor. It has been reported that the folate receptor β,the nonepithelial isoform of the folate receptor, is expressed onactivated (but not resting) synovial macrophages. Thus, folate receptorsare expressed on a subset of macrophages (i.e., activated macrophages).Folate receptors of the β isoform are also found on activated monocytes.

Accordingly, the present invention relates to the development ofvitamin-targeted therapeutics, such as folate-targeted therapeutics, totreat inflammation. The folate conjugates described herein can be usedto treat inflammatory diseases by targeting inflammatory cells thatoverexpress the folate receptor.

SUMMARY OF THE INVENTION

It has been discovered that therapeutic agents, diagnostic agents, andimaging agents may be conjugated to other compounds to control or altertheir behavior, biodistribution, metabolism, and/or clearance in vivo.In one illustrative embodiment of the invention, conjugates of compoundsare described that include a hydrophilic spacer linker. In one aspect,conjugates of compounds are described that include both a hydrophilicspacer linker and a targeting ligand. Illustrative of such conjugatesare compounds of the following formula described herein

B-L-A

wherein B is a receptor binding ligand that binds to a target cellreceptor, L is a linker that comprises one or more hydrophilic spacerlinkers, and A is a therapeutic agent (e.g. a drug) that is desirablydelivered to the cell.

In one variation, the linker L does not include a releasable linker. Inanother variation, the linker L includes a releasable linker. In anotherembodiment, at least one of the hydrophilic spacer linkers is formedfrom or includes at least one carbohydrate. In one variation, thecarbohydrate forms part of the linker chain connecting B and A. Inanother variation, the carbohydrate forms part of a side chain attachedto the linker chain connecting B and A. In one variation, the linker isa polyvalent linker. In another variation, the linker is a bivalentlinker.

It is appreciated that in each of the above embodiments, more than onereceptor binding ligand B may be attached to the linkers describedherein. It is further appreciated that more than one therapeutic agent Amay be attached to the linkers described herein. Such multi-ligandand/or multi-drug conjugates are also described herein, where the linker(L) comprises a hydrophilic spacer linker.

In another embodiment, compounds are described herein that have reduceduptake by the liver and are less likely to be cleared by the liver. Inone aspect, such compounds are preferentially cleared by the renalprocesses as compared to hepatic processes.

The therapeutic agent or therapeutic agents A include therapeutic drugsand any other compound that is desirably or advantageously delivered toa cell by targeting a cell receptor. Illustrative drugs includecytotoxic drugs, anti-inflammatory agents, and the like.

In the embodiments of compounds, compositions, and methods describedherein, the cells that may be targeted with the therapeutic agents Ainclude cells that cause inflammation, such as activated monocytes,activated macrophages, and other inflammatory cells. The targeting ofthe cell is accomplished by the appropriate selection of a receptorbinding ligand B. It is appreciated that selective or specific targetingof a cell in vivo may be accomplished by selecting a receptor that ispreferentially expressed or overexpressed by the target cell.Illustratively, the target cell preferentially expresses oroverexpresses a vitamin receptor, such as a folate receptor.

In another embodiment, the conjugates described herein are included inpharmaceutical compositions in amounts effective to treat disease statesassociated with pathogenic populations of cells, such as cellsassociated with inflammation.

In another embodiment, the conjugates described herein, andpharmaceutical compositions containing them are used in methods fortreating diseases and disease states associated with pathogenicpopulations of cells, such as cells associated with inflammation.

In another embodiment, a method for treating a patient with aninflammatory disease, the method comprising the step of administering tothe patient a composition comprising a drug delivery conjugate of theformula

BL(A¹)(A²)_(m)

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

m is 0 or 1;

B is a folate;

L is a linker that comprises one or more hydrophilic spacer linkers;

A¹ is an antifolate; and

A² has the formula

wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug forming group, and C(O)R^(D), where R^(D) is ineach instance independently selected from the group consisting ofhydrogen, and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted is described.

In another embodiment, a pharmaceutical composition comprising a drugdelivery conjugate of the formula

BL(A¹)(A²)_(m)

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

m, B, L, A¹, A², Y^(A), R^(A), R^(B), R^(C), and R^(D) are as describedherein.

In any of the preceding embodiments, the antifolate can be aminopterin,or an analog, derivative, or conjugate thereof.

In another embodiment, a method for treating a patient with aninflammatory disease, the method comprising the step of administering tothe patient a composition comprising a drug delivery conjugate of theformula

B-L-A³

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

B is a folate;

L is a linker that comprises one or more hydrophilic spacer linkers; and

A³ has the formula

wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug forming group, and C(O)R^(D), where R^(D) is ineach instance independently selected from the group consisting ofhydrogen, and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted is described.

In another embodiment, a pharmaceutical composition comprising a drugdelivery conjugate of the formula

B-L-A³

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

m, B, L, A³, Y^(A), R^(A), R^(B), R^(C), and R^(D) are as describedherein.

In another embodiment, a kit comprising a sterile vial, the compositionof any one of the preceding embodiments, and instructions for usedescribing use of the composition for treating a patient with aninflammatory disease is described.

In another embodiment, is described a kit comprising a sterile vial, acomposition comprising the compound as a lyophilized solid of any one ofthe preceding embodiments, and instructions describing use of thecomposition for treating a patient with an inflammatory disease, whereinthe vial is an amber glass vial with a rubber stopper and an aluminumtear-off seal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Measurement of DHFR activity in the lysate of RAW264.7 cellsafter no treatment, treatment with EC0746 (1 hour treatment followed by24 hours in treatment-free medium), treatment with EC0746 plus excessfolic acid (EC0746/FA, 1 hour treatment followed by 24 hours intreatment-free medium), treatment with aminopterin (AMT, 24 hourtreatment), treatment with methotrexate (MXT, 24 hour treatment), andtreatment with folic acid (FA, 10 μM).

FIG. 2A—Viability of RAW264.7 cells, measured using the XTT assay,treated with EC0746 (EC₅₀ 0.33 nM, 2 hour treatment followed by 72 hoursin treatment-free medium) and treated with EC0746 and excess folic acid(EC0746/FA, 2 hour treatment followed by 72 hours in treatment-freemedium). FIG. 2B—Inhibition of LPS-stimulated TNF-α production inRAW264.7 cells treated with EC0746 (EC₅₀ 1.43 nM, 2 hour treatmentfollowed by 72 hours in treatment-free medium) and treated with EC0746and excess folic acid (EC0746/FA, 2 hour treatment followed by 72 hoursin treatment-free medium).

FIG. 3. Inhibition of LPS-stimulated cytokine production (LPS (5 ug/mL),IFN-γ (100 ng/mL) for 24 hours) in thioglycolate-elicited macrophages.Compounds at 100 nM with 100xs FA (2 hour treatment followed by 72 hoursin fresh medium, unstimulated cell; stimulated cells without treatment(LPS/IFN-γ), treatment with excess folic acid, treatment with EC0746(EC0746), treatment with EC0746 and excess folic acid (EC0746/FA),treatment with methotrexate, and treatment with aminopterin. Relativefolate receptor binding affinities (folic acid defined as 1.00) EC0746,0.50; aminopterin, 0.004; and methotrexate, 0.018.

FIG. 4A—Arthritis scores for a) untreated animals with adjuvant inducedarthritis and b) animals treated with EC0746 (500 nmol/kg). FIG.4B—Percentage change in body weight measured for a) untreated animalswith adjuvant induced arthritis and b) animals treated using EC0746 (500nmol/kg).

FIG. 5A—Paw weights measured after the end of the treatment period forhealthy animals, untreated animals with adjuvant induced arthritis andb) animals treated with EC0746 (500 nmol/kg). FIG. 5B—Spleen weightsmeasured after the end of the treatment period for healthy animals,untreated animals with adjuvant induced arthritis and b) animals treatedwith EC0746 (500 nmol/kg).

FIG. 6A—Photographs of the hind paws of a healthy control animal, anuntreated animal with adjuvant induced arthritis, and an animal treatedwith EC0746 (500 nmol/kg); FIG. 6B—X-rays of the hind paws of a healthycontrol animal, an untreated animal with adjuvant induced arthritis, andan animal treated with EC0746 (500 nmol/kg).

FIG. 7A—Average arthritis scores measured during the treatment periodfor a) untreated animals with adjuvant induced arthritis, b) animalstreated bi-weekly with EC0746, 300 nmole/kg; c) with methotrexate, 300nmole/kg; and d) healthy untreated animals. Treatments administered onthe days indicated with the arrows. FIG. 7B—Percentage weight changemeasured during the treatment period for a) untreated animals withadjuvant induced arthritis, b) animals treated bi-weekly with EC0746,300 nmole/kg; c) animals treated bi-weekly with methotrexate, 300nmole/kg; and d) healthy untreated animals.

FIG. 8A—Paw weights measured at the end of the treatment period forhealthy untreated animals, untreated arthritic animals, animals treatedbi-weekly with EC0746 (300 nmole/kg), and animals treated bi-weekly withmethotrexate (300 nmole/kg). FIG. 8B—Spleen weights measured at the endof the treatment period for healthy untreated animals, untreatedarthritic animals, animals treated bi-weekly with EC0746 (300 nmole/kg),and animals treated bi-weekly with methotrexate (300 nmole/kg).

FIG. 9. Viability of RAW264.7 macrophage cells treated in mediacontaining the test compound or compounds for 2 hours at theconcentration shown in the graph, followed by 72 hours in fresh mediumwithout the test compound(s); a) EC0746; b) EC0746+excess folic acid; c)EC0808 (a D-aminopterin diastereoisomer of EC0746); and EC0808+excessfolic acid.

FIG. 10. Arthritis score measured for a) untreated arthritic animals; b)animals treated bi-weekly with methotrexate (500 nmole/kg), and c)animals treated bi-weekly with EC0808 (500 nmole/kg).

FIG. 11A—Arthritis score measured for a) untreated arthritic animals; b)animals treated bi-weekly with EC0746 (500 nmole/kg), c) animals treatedbi-weekly with EC0746 (500 nmole/kg)+300-fold excess of Re-EC0589), andd) healthy untreated animals. FIG. 11B—The percentage body weight changemeasured for the same animals used to obtain the arthritis scores inFIG. 11A. Percentage weight change measured for a) untreated arthriticanimals; b) animals treated bi-weekly with EC0746 (500 nmole/kg), c)animals treated bi-weekly with EC0746 (500 nmole/kg)+300-fold excess ofRe-EC0589), and d) healthy untreated animals.

FIG. 11C—Arthritis score measured for a) healthy animals, b) untreatedarthritic animals; c) animals treated bi-weekly with EC0746 (250nmole/kg), d) animals treated bi-weekly with EC0746 (250nmole/kg)+500-fold excess of EC0923), and e) animals treated biweeklywith EC0923 alone.

FIG. 12A—Arthritis score measured for a) untreated arthritic animals; b)animals treated bi-weekly with methotrexate (500 nmole/kg), c) animalstreated bi-weekly with methotrexate (500 nmole/kg)+300-fold excess ofRe-EC20), and d) arthritic animals treated with Re-EC20. FIG. 12B—Thepercentage body weight change measured for the same animals used toobtain the arthritis scores in FIG. 12A. The percentage weight changemeasured for a) Untreated arthritic animals; b) animals treatedbi-weekly with methotrexate (500 nmole/kg), c) animals treated bi-weeklywith methotrexate (500 nmole/kg)+300-fold excess of Re-EC20), and d)arthritic animals treated with Re-EC20.

FIG. 13A—The average arthritis score measured using a two-dosage leveltreatment schedule, a 500 nmole/kg dose was administered on days 10 and15 post-arthritis induction and a 100 nmole/kg dose was administered onday 11-14 and 16-19. a) untreated arthritic animals (collagen-inducedarthritis, rats induced treatment with 500 μg Type II collagen/Freund'scomplete adjuvant at day 0 followed by 500 μg Type II collagen/Freund'sincomplete adjuvant at day 7, b) animals treated with EC0746, and c)animals treated with methotrexate. FIG. 13B—The percentage weight changemeasured for the animals used to obtain the arthritis scores shown inFIG. 13A. a) Untreated arthritic animals, b) animals treated withEC0746, and c) animals treated with methotrexate.

FIG. 14A—Plasma concentrations measured after a single subcutaneous doseof EC0746 (500 nmole/kg). a) EC0746, b) EC0470 (aminopteringamma-hydrazide), and c) aminopterin. FIG. 14B—The C_(max) for EC0746 of321 nmole/L is reached at 30 minutes post injection. The C_(m)ax forfree drug (aminopterin+aminopterin hydrazide) of 34 nmole/L is reachedat 60 minutes. C_(max) for free drug is 9.6% of the total dose. The AreaUnder Curve value for EC0746 is 32.5 nmole-min/mL, the Area Under Curvevalue for free drug is 7.3 nmole-min/mL (18% of the total).

FIG. 15. Plasma concentrations measured after a single subcutaneous doseof aminopterin (500 nmole/kg). The maximum plasma concentration wasmeasured at 30 minutes post administration.

FIG. 16A—Percentage body weight change measured for animals aftersubcutaneous administration of the indicated dose of aminopterinbiweekly for 2 weeks a) control, 0 nmole/kg, b) 100 nmole/kg, and c) 50nmole/kg. FIG. 16B—Percentage body weight change measured for animalsafter subcutaneous administration of the indicated dose of treatmentcompound biweekly for 2 weeks a) control, no treatment, b) 100 nmole/kgaminopterin, and c) 50 nmole/kg aminopterin, d) 500 nmole/kg EC0746, ore) 2000 nmole/kg EC0746.

FIG. 17A—Viability of RAW264.7 cells, measured using the XTT assay,treated with EC0932 (2 hour treatment followed by 72 hours intreatment-free medium) and treated with EC0932 and excess folic acid(EC0932/FA, 2 hour treatment followed by 72 hours in treatment-freemedium). FIG. 17B—Inhibition of LPS-stimulated TNF-a production inRAW264.7 cells treated with EC0932 (2 hour treatment followed by 72hours in treatment-free medium) and treated with EC0932 and excess folicacid (EC0932/FA, 2 hour treatment followed by 72 hours in treatment-freemedium).

FIG. 18A—A Western blot showing inhibition of mTOR signaling inLPS/IFN-γ stimulated RAW264.7 cells. Comparison between unstimulatedcells, untreated stimulated cells, stimulated cells treated with an mTORinhibitor (everolimus), stimulated cells treated with an antifolate(aminopterin), stimulated cells treated with EC0932, stimulated cellstreated with EC0932 plus excess competitor (EC0823), and stimulatedcells treated excess competitor (EC0823) alone. Treatments with 100 nMcompound in cell medium for 2 hours followed by fresh untreated medium.FIG. 18B—measurement treatment response on pRPS6.

FIG. 19A—Arthritis score measured for a) untreated animals with inducedarthritis, b) healthy untreated animals, c) animals treated with EC0932(250 nmole/kg, biweekly on indicated days), and d) animals treated withEC0932 and a 500-fold excess of EC0923 (250 nmole/kg, biweekly, onindicated days). FIG. 19B—Percentage change in body weight measured forthe animals used to obtain the arthritis scores shown in FIG. 19A; a)untreated animals with induced arthritis, b) healthy untreated animals,c) animals treated with EC0932 (250 nmole/kg, biweekly on indicateddays), and d) animals treated with EC0932 and a 500-fold excess ofEC0923 (250 nmole/kg, biweekly, on indicated days).

FIG. 20A—Paw weights measured after the completion of the treatment andobservation period used to obtain the data in FIG. 19 for a) untreatedanimals with induced arthritis, b) healthy untreated animals, c) animalstreated with EC0932 (250 nmole/kg, biweekly on indicated days), and d)animals treated with EC0932 and a 500-fold excess of EC0923 (250nmole/kg, biweekly, on indicated days). FIG. 20B—Spleen weights measuredafter the completion of the treatment and observation period used toobtain the data in FIG. 20A for a) untreated animals with inducedarthritis, b) healthy untreated animals, c) animals treated with EC0932(250 nmole/kg, biweekly on indicated days), and d) animals treated withEC0932 and a 500-fold excess of EC0923 (250 nmole/kg, biweekly, onindicated days).

FIG. 21A—Arthritis score measured for a) untreated animals with inducedarthritis, b) healthy untreated animals, c) animals treated with EC0894(500 nmole/kg, biweekly), and d) animals treated with EC0828 (250nmole/kg, biweekly). FIG. 21B—Percentage change in body weight measuredfor the animals used to obtain the arthritis scores shown in FIG. 21A;a) untreated animals with induced arthritis, b) healthy untreatedanimals, c) animals treated with EC0894 (500 nmole/kg, biweekly), and d)animals treated with EC0828 (250 nmole/kg, biweekly).

FIG. 22A—Paw weight measured after completion of the treatment andobservation period used to obtain the data in FIG. 21 for a) untreatedanimals with induced arthritis, b) healthy untreated animals, c) animalstreated with EC0894 (500 nmole/kg, biweekly), and d) animals treatedwith EC0828 (250 nmole/kg, biweekly). FIG. 22B—Spleen weight measuredafter completion of the treatment and observation period used to obtainthe data in FIG. 21 for a) untreated animals with induced arthritis, b)healthy untreated animals, c) animals treated with EC0894 (500 nmole/kg,biweekly), and d) animals treated with EC0828 (250 nmole/kg, biweekly).

FIG. 23A—Arthritis score measured for a) untreated animals with inducedarthritis, b) healthy untreated animals, c) animals treated with EC0565(500 nmole/kg, biweekly), and d) animals treated with EC0828 (250nmole/kg, biweekly). FIG. 23B—Percentage change in body weight measuredfor the animals used to obtain the arthritis scores shown in FIG. 23A;a) untreated animals with induced arthritis, b) healthy untreatedanimals, c) animals treated with EC0894 (500 nmole/kg, biweekly), and d)animals treated with EC0828 (250 nmole/kg, biweekly).

FIG. 24A—Paw weight measured after completion of the treatment andobservation period used to obtain the data in FIG. 23 for a) untreatedanimals with induced arthritis, b) healthy untreated animals, c) animalstreated with EC0565 (500 nmole/kg, biweekly), and d) animals treatedwith EC0828 (250 nmole/kg, biweekly). FIG. 24B—Spleen weight measuredafter completion of the treatment and observation period used to obtainthe data in FIG. 23 for a) untreated animals with induced arthritis, b)healthy untreated animals, c) animals treated with EC0894 (500 nmole/kg,biweekly), and d) animals treated with EC0828 (250 nmole/kg, biweekly).

FIGS. 25A-D—Relative affinities of EC0746 (FIG. 25A), aminopterin (AMT)(FIG. 25B), methotrexate (MTX) (FIG. 25C), and EC0932 (FIG. 25D),compared to folic acid (FA), set as 1, to folate receptors (FR-α) of KBcells. FIG. 25E—In vitro inhibition of DHFR in FR-positive RAW264.7cells: Untreated control; EC0746 (2 h pulse of 100 nm, followed by 22 h“chase” in drug free medium); EC0746 with excess FA (2 h pulse of 100 nmEC0746 with 100-fold excess folic acid (folate competition), followed by22 h “chase”); AMT (2 h pulse of 100 nm, followed by 22 h “chase” indrug free medium); MTX (2 h pulse of 100 nm, followed by 22 h “chase” indrug free medium); FA alone (2 h pulse of 10 μm, followed by 22 h“chase” in drug free medium).

FIG. 25F—Relative affinities of EC0746, aminopterin (AMT), andmethotrexate (MTX) compared to folic acid (FA), set as 1, to folatereceptors (FR-β) of CHO cells. a. folic acid (relative affinity set to1.0), b. EC0746 (relative affinity 0.270), c. aminopterin (relativeaffinity 0.004), and d. methotrexate (relative affinity 0.005).

FIG. 26A, B, C, D, E, F, G, H, I. Anti-proliferative Effect on RAW264.7cells: FIG. 26A—Viability of RAW264.7 cells, measured using the XTTassay, LPS (100 ng/mL) added at 4 h before end of incubation tostimulate cytokine production, treated with EC0746 (EC₅₀ about 0.3 nM, 2hour treatment followed by 70 h “chase” in drug-free medium) and treatedwith EC0746 and excess folic acid (EC0746/FA, 2 hour treatment followedby 70 h “chase” in drug-free medium). FIG. 26B—TNF-α production fromcells treated as in part a, upon LPS exposure (ED₅₀ about 1.6 nM). Flowcytometric analysis (FACS) with propidium iodide (PI) staining of thecell cycle of the cells of part a for Untreated (FIG. 26C),EC0746-treated (FIG. 26D) and EC0746/FA-treated cells (FIG. 26E). FIG.26F—Cell cycle distribution for Untreated, EC0746 treated and EC0746/FAtreated cells. FIG. 26G —Western blot analysis of cell-cycledistribution (d) and PCNA expression (e) on whole cell lysates using aPCNA-specific monoclonal antibody for Untreated (UTC), EC0746-treated(EC0746-FA) and EC0746/FA (EC0746+FA)-treated cells.

FIGS. 26H, I—Anti-proliferative Effect on RAW264.7 cells: FIG.26H—Viability of RAW264.7 cells, measured using the XTT assay, LPS (100ng/mL) added at 4 h before end of incubation to stimulate cytokineproduction, 2 hour treatment followed by 70 h “chase” in drug-freemedium). Comparison of EC0746, EC0746 with excess folic acid(EC0746/FA), aminopterin (AMT), and methotrexate (MTX). FIG. 26I—TNF-αproduction from cells treated as in part a upon LPS exposure. Comparisonof EC0746, EC0746 with excess folic acid (EC0746/FA), aminopterin (AMT),and methotrexate (MTX).

FIG. 27A, B, C. EC0746 Modulation of Cytokine Responses inThioglycollate-elicited Macrophages (TG-macs). FIGS. 27A, B—Rat cytokineantibody array and plotted results for cytokines/chemokines (FIG. 27C)for rat TG-macs untreated (LPS) or with indicated treatment of 100 nM oftreatment for 2 h plus a 70 h chase, and with addition at 24 h prior toend of incubation of LPS (5 μg/mL) and IFN-γ (100 ng/mL) for EC0746,EC0746 plus 100-fold excess FA (EC0746/FA), folic acid alone (FA),aminopterin (AMT), and methotrexatre (MTX), respectively, as to IL-1β,IL-Ira, IL-10, MIP-Iα, TNF-α, VEGF, CINC-2a/b, CINC-3, sICAM, LIX,L-selectin, and MIG.

FIG. 28A, B, C. Amelioration of Systemic Inflammation in Rats withAdjuvant Induced Arthritis (AIA). FIG. 28A—Arthritis score with time indays since first treatment; FIG. 28B—% Change in body weight with timein days since first treatment; and FIG. 28C—% Increase in weight of Pawsand Spleen for Healthy, Arthritic (onset), EC0746-treated (onset),Arthritic (established) and EC0746-treated (established) AIA rats.

FIG. 29A, B, C, D, E, F. Anti-arthritic activity in Rats with AdjuvantInduced Arthritis (AIA). FIG. 29A—Arthritis score with time in dayssince first treatment; FIG. 29B—% Change in body weight with time indays since first treatment; FIG. 29C—% Increase in weight of Paws; FIG.29D—% Increase in weight of Spleen; FIG. 29E—radiograph of hind paw; andFIG. 29F—Radiographic score for Healthy (where indicated), ArthriticControl (untreated), and animals treated with EC0746, EC0746+Competitor,Competitor alone, methotrexate (MTX), or Enbrel (etanercept) in AIArats, with EC0923 as the folate-containing competitor.

FIG. 30A, B, C, D. Histology of Rats with Adjuvant Induced Arthritis(AIA). FIG. 30A—Histology scores for Inflammation, Pannus, Cartligedamage and Bone Resorption; FIG. 30B—Sum histology scores; FIG. 30C—Meanpaw thickness (rpm) and FIG. 30D—images from paws for Healthy (whereindicated), Arthritic Control (untreated), and animals treated withEC0746, EC0746+Competitor, Competitor alone, methotrexate (MTX), orEnbrel in AIA rats, with EC0923 as the folate-containing competitor.

FIG. 31A, B, C. Anti-arthritic activity in Rats with Adjuvant InducedArthritis (AIA). FIG. 31A—Arthritis score with time in days since firsttreatment; FIG. 31B—% Change in body weight with time in days sincearthritis induction; FIG. 31C—% Increase in weight of Paws and Spleenfor Healthy (where indicated), Arthritic Control (untreated), andanimals treated with methotrexate (MTX) or MTX+Competitor in AIA rats,with EC0923 as the folate-containing competitor.

FIG. 32A, B. Effects of potential EC0746 Metabolites on RAW264.7 cells.The effects of the potential EC0746 metabolites aminopterin (AMT) andAMT hydrazide (EC0470) in 72 h incubations of RAW264.7 macrophages areshown for: FIG. 32A—Cell proliferation in the XTT assay, and FIG.32B—LPS-stimulated TNF-α production.

FIG. 33A, B, C. Pharmacokinetics of EC0746 and potential metabolitesaminopterin (AMT) and AMT hydrazide (EC0470) in Rats. FIG. 33A—Plasmaconcentrations (nmol/L) of EC0746, AMT and AMT hydrazide followingsingle subcutaneous EC0746 (500 nmol/kg) administration. FIG. 33B—Plasmaconcentrations (nmol/L) of AMT following single subcutaneous AMT (500nmol/kg) administration. FIG. 33C—Pharmacokinetic analysis of theresults of FIGS. 33A, B.

FIG. 34. An animal model for autoimmune disease uveitis. Rats wereimmunized with a bovine S antigen peptide emulsified with Freund'sincomplete adjuvant containing M. Tuberculosi and boosted with pertussistoxin.

FIG. 35. Uveitis total scores (both eyes) for animals treated with 500nmol/kg EC0746 every other day starting on day 7 after EAU induction(open circles) or from untreated animals (closed circles).

FIG. 36A, B, C. Representative photographs of rat eyes were taken on day15. FIG. 36A—A photograph of an eye from an untreated animal withexperimental autoimmune uveitis (EAU). FIG. 36B—A photograph of an eyefrom an animal with EAU treated with EC0746 every other day starting onday 7 after EAU induction. FIG. 36C—A photograph of an eye from ahealthy rat.

FIG. 37. The effect of EC0746 treatment on protein levels in the aqueoushumor. The protein levels (mg/mL) at day 19 in the aqueous humor in theanterior portion of the eye are shown for the left and right eye of eachtested animal. Animals 1-5 were untreated after induction of the EAU.Animals 6-9 were treated with EC0746 every other day starting on day 7after EAU induction. On the far right of the chart, the total of theprotein levels in the aqueous humor samples pooled from both eyes of anuntreated, healthy animal.

FIG. 38A shows that EC0565 induces inhibition of RPS6 in RAW264.7 cells(1 h pulse/6 h chase), where UTC=Control (untreated cells); EC0565=(100nM) and EC0565+x.s. EC17=treatment plus an excess amount of anon-cytotoxic folate conjugate; FIG. 38B shows that EC0565 inducesinhibition of RPS6 in TG-elicited macrophages (1 h pulse/6 h chase), ina dose dependent manner, where UTC=Control (untreated cells);EC0565=treatment (10 nmol, 30 nmol, 100 nmol); EC0565+x.s.folate=treatment (10 nmol, 30 nmol, 100 nmol) plus an excess of a folicacid conjugate (100 μmole); FAC=treatment with folic acid and 0 nmol ofEC0565; and Everolimus=treatment with unconjugated everolimus (10 nmol,100 nmol); FIG. 38C shows that EC0565 induces inhibition of RPS6 inarthitic macrophages (1 h pulse/6 h chase), in a dose dependent manner,where UTC=Control (untreated cells); EC0565=treatment (1 nmol, 10 nmoland 30 nmol); EC0565+excess folate=treatment (1 nmol, 10 nmol and 30nmol) plus an excess of a folic acid (100 μmole); and FAC=treatment withfolic acid and 0 nmol of EC0565.

FIG. 39 compares the arthritis score of animals treated with biweeklyinjections of 500 nmol/kg of EC0565 (d) and biweekly injections of 500nmol/kg of unconjugated everolimus (c) with healthy controls (a) anduntreated animals (b).

FIG. 40 shows the percentage weight change observed for the same animalsused to generate the data shown in FIG. 39. (a) Untreated animals, (b)treated with biweekly injections of 500 nmol/kg of EC0565, (c) untreatedhealthy control animals, and (d) treated with biweekly injections of 500nmol/kg of unconjugated everolimus.

FIG. 41 shows the paw weight data (localized disease) for the animals atthe end of the treatment period for each of the treatment groupsdescribed in FIGS. 39 and 40.

FIG. 42 shows the spleen weight data (systemic disease) for the animalsat the end of the treatment period for each of the treatment groupsdescribed in FIGS. 39 and 40.

FIG. 43 shows the radiographic analysis of soft tissue and bone damagein the hind paws of animals treated with EC0565 (500 nmol/kg),Everolimus (500 nmol/kg), methotrexate (MTX, 190 nmol/kg), untreatedanimals with adjuvant induced arthritis, and untreated, healthy controlanimals.

FIG. 44A compares the arthritis score of animals treated with biweeklyinjections of 500 nmol/kg of EC0565 (d) and biweekly injections of 500nmol/kg of unconjugated everolimus (c) with healthy controls (b) anduntreated animals (a). FIG. 44B shows the percentage weight changeobserved for the treatment groups shown in FIG. 44A. FIG. 44C shows thepaw weight data (localized disease) for the animals at the end of thetreatment period for each of the treatment groups described in FIGS.44A, B. FIG. 44D shows the spleen weight data (systemic disease) for theanimals at the end of the treatment period for each of the treatmentgroups described in FIGS. 44A, B.

FIG. 45 shows that EC0565 induces dose-responsive inhibition of theproduction of pRPS6 and p70S6K in KB cells (1 h pulse/4 h chase) using a30 min camera exposure, where C=Control (untreated cells); FAC=Folicacid control (100 μM).

FIG. 46A. Arthritis scores for AIA rats. a) untreated animals; b)healthy animals; c) EC0565, subcutaneously (s.c.), 500 nmol/kg, tiw; d)everolimus, oral, 500 nmol/kg, tiw; e) etanercept, s.c. 10 mg/kg, q3d;and e) methotrexate (MTX), oral, 250 nmol/kg, biw).

FIG. 46B. Weight change for AIA rats. a) untreated animals; b) healthyanimals; c) EC0565, subcutaneously (s.c.), 500 nmol/kg, tiw; d)everolimus, oral, 500 nmol/kg, tiw; e) etanercept, s.c. 10 mg/kg, q3d;and e) methotrexate (MTX), oral, 250 nmol/kg, biw).

FIG. 46C. Paw Weights (a measure of swelling) for AIA rats. healthyanimals; untreated controls; EC0565, subcutaneously (s.c.), 500 nmol/kg,tiw; everolimus, oral, 500 nmol/kg, tiw; etanercept, s.c. 10 mg/kg, q3d;and methotrexate (MTX), oral, 250 nmol/kg, biw).

FIG. 46D. Spleen Weights for AIA rats. Healthy animals; untreatedcontrols; EC0565, subcutaneously (s.c.), 500 nmol/kg, tiw; everolimus,oral, 500 nmol/kg, tiw; Enbrel (etanercept), s.c. 10 mg/kg, q3d; andmethotrexate (MTX), oral, 250 nmol/kg, biw).

FIG. 47. Radiographic analysis of hind paws of AIA rats. Healthyanimals; untreated controls; EC0565, subcutaneously (s.c.), 500 nmol/kg,tiw; everolimus, oral, 500 nmol/kg, tiw; Enbrel (etanercept), s.c. 10mg/kg, q3d; and methotrexate (MTX), oral, 250 nmol/kg, biw.

FIG. 48A—Histological study of AIA rats. Scores for inflammation, pannusformation, cartilage damage, and bone resorption are shown for untreatedanimals (untreated control); everolimus, oral, 500 nmol/kg, tiw;methotrexate (MTX), oral, 250 nmol/kg, biw; EC0565, subcutaneously(s.c.), 500 nmol/kg, tiw; and etanercept, s.c. 10 mg/kg, q3d. FIG.48B—The sum of the scores shown in panel a for each treatment. FIG.48C—The measured paw thickness for each treatment shown in FIG. 48A.

FIG. 49 shows representative photomicrographs (16×) of the ankle closestto the mean summed score for each treatment group.

FIG. 50A shows the amount of paw edema for healthy or AIA rats. Healthyrats, no induces arthritis; arthritis (untreated AIA rats); treated withEC0565 100 nmol/kg/dose, twice/week; 500 nmol/kg/dose, twice/week; 1000nmol/kg, twice/week; and 1000 nmol/kg/dose, once/week. FIG. 50B showsthe change in spleen weight of for healthy or AIA rats. Healthy rats, noinduces arthritis; arthritis (untreated AIA rats); treated with EC0565100 nmol/kg/dose, twice/week; 500 nmol/kg/dose, twice/week; 1000nmol/kg, twice/week; and 1000 nmol/kg/dose, once/week.

FIG. 51A shows the average arthritis score for treated rats withcollagen-induced arthritis (CIA). a) untreated CIA animals; b) ECO565,1000 nmol/kg/dose, tiw; and c) everolimus, 1000 nmol/kg/dose, tiw.

FIG. 51B shows the average weight change for animals in treat as in FIG.51A. a) untreated CIA animals; b) ECO565, 1000 nmol/kg/dose, tiw; and c)everolimus, 1000 nmol/kg/dose, tiw.

FIG. 52A shows the plasma concentration of EC0565 and everolimus overtime after a 2 mmol/kg intravenous dose of EC0565. FIG. 52B shows theplasma concentration of EC0565 and everolimus over time after a 2mmol/kg subcutaneous dose of EC0565. FIG. 52C shows a comparison of theplasma concentration of EC0565 given subcutaneously or intravenously.

FIG. 53A shows the effect on Proliferating Cell Nuclear Antigen (PCNA)in synchronized FR-positive murine macrophage-like RAW264.7 cellstreated with media as measured by Western blot analysis on whole celllysates using a monoclonal antibody specific for PCNA, a) EC0565 (1 nM,10 nM, 100 nM, and 1000 nM); b) EC0565 (1 nM, 10 nM, 100 nM, and 1000nM) in the presence of XS EC17 (a folate receptor binding competitor);c) EC17 alone (1 nM, 10 nM, 100 nM, and 1000 nM); and d) everolimus (1nM, 10 nM, 100 nM, and 1000 nM).

FIG. 53B shows the pixel density measurements for the images shown inFIG. 53A a) EC0565 (1 nM, 10 nM, 100 nM, and 1000 nM); b) EC0565 (1 nM,10 nM, 100 nM, and 1000 nM) in the presence of XS EC17 (a folatereceptor binding competitor); c) EC17 alone (1 nM, 10 nM, 100 nM, and1000 nM); and d) everolimus (1 nM, 10 nM, 100 nM, and 1000 nM).

FIG. 53C shows a plot of percent control of PCNA data shown in FIG. 53Bfor EC0565 (1 nM, 10 nM, 100 nM, and 1000 nM); EC0565 (1 nM, 10 nM, 100nM, and 1000 nM) in the presence of XS EC17 and for everolimus (1 nM, 10nM, 100 nM, and 1000 nM).

DETAILED DESCRIPTION

Drug delivery conjugates are described herein consisting of a receptorbinding ligand (B), a linker (L) comprising one or more hydrophilicspacer linkers, and a therapeutic agent (A), e.g. a drug, that isdesirably delivered to a cell. The receptor binding ligand (B) iscovalently attached to the linker (L), and the therapeutic agent (A), oran analog or derivative thereof, is also covalently attached to thelinker (L). It is to be understood that the therapeutic agent (A)includes analogs and derivatives thereof that are attached to the linker(L). The linker (L) comprises one or more spacer linkers and/orreleasable linkers, and combinations thereof, in any order. In onevariation, releasable linkers, and optional spacer linkers arecovalently bonded to each other to form the linker. In anothervariation, a releasable linker is directly attached to the therapeuticagent (A), or analog or derivative thereof. In another variation, areleasable linker is directly attached to the receptor binding ligand(B). In another variation, either or both the receptor binding ligand(B) and the therapeutic agent (A), or analog or derivative thereof, isattached to a releasable linker through one or more spacer linkers. Inanother variation, each of the receptor binding ligand (B) and thetherapeutic agent (A), or analog or derivative thereof, is attached to areleasable linker, each of which may be directly attached to each other,or covalently attached through one or more spacer linkers.

From the foregoing, it should be appreciated that the arrangement of thereceptor binding ligand (B), and the therapeutic agent (A), or analog orderivative thereof, and the various releasable and optional spacerlinkers may be varied widely. In one aspect, the receptor binding ligand(B), and the therapeutic agent (A), or analog or derivative thereof, andthe various releasable and optional spacer linkers are attached to eachother through heteroatoms, such as nitrogen, oxygen, sulfur, phosphorus,silicon, and the like. In variations, the heteroatoms, excluding oxygen,may be in various states of oxidation, such as N(OH), S(O), S(O)₂, P(O),P(O)₂, P(O)₃, and the like. In other variation, the heteroatoms may begrouped to form divalent radicals, such as for example hydroxylamines,hydrazines, hydrazones, sulfonates, phosphinates, phosphonates, and thelike, including radicals of the formulae —(NHR¹NHR²)—, —SO—, —(SO₂)—,and —N(R³)O—, wherein R¹, R², and R³ are each independently selectedfrom hydrogen, alkyl, aryl, arylalkyl, substituted aryl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, and alkoxyalkyl. In onevariation, the linker (L) is a polyvalent linker. In another variation,more than one receptor binding ligand (B) is attached to the polyvalentlinker. In another variation, more than one therapeutic agent (A) isattached to the polyvalent linker. In another variation, more than onereceptor binding ligand (B) and more than one therapeutic agent (A) isattached to the polyvalent linker.

In one embodiment, the receptor binding ligand (B) is a vitamin receptorbinding ligand such as a vitamin, or an analog or a derivative thereof,capable of binding to vitamin receptors. In another embodiment, thereceptor binding ligand (B) is a vitamin, or analog or derivativethereof, attached to a releasable linker which is attached to the drugthrough a linker (L) that is formed from one or more spacer linkersand/or releasable linkers and/or hydrophilic spacer linkers. In onevariation, both the therapeutic agent (A) and the vitamin, or analog orderivative thereof, can each be attached to spacer linkers, where thespacer linkers are attached to each other through one or more releasablelinkers. In addition, both the therapeutic agent (A) and the vitamin, oranalog or derivative thereof, can each be attached to one or morereleasable linkers, where the releasable linkers are attached to eachother or through a spacer linker. Each of these radicals may beconnected through existing or additional heteroatoms on the receptorbinding ligand (B), therapeutic agent (A), or releasable, hydrophilicspacer, or additional spacer linker.

The binding site for the receptor binding ligand (B) can includereceptors for any binding ligand (B), or a derivative or analog thereof,capable of specifically binding to a receptor wherein the receptor orother protein is uniquely expressed, overexpressed, or preferentiallyexpressed by a population of pathogenic cells. A surface-presentedprotein uniquely expressed, overexpressed, or preferentially expressedby the pathogenic cells is typically a receptor that is either notpresent or present at lower concentrations on non-pathogenic cellsproviding a means for selective elimination of the pathogenic cells. Thedrug delivery conjugates may be capable of high affinity binding toreceptors on activated macrophages, monocytes, or other inflammatorycells. The high affinity binding can be inherent to the binding ligandor the binding affinity can be enhanced by the use of a chemicallymodified ligand (e.g., an analog or a derivative of a vitamin).

The drug delivery conjugates described herein can be formed from, forexample, a wide variety of vitamins or receptor-binding vitaminanalogs/derivatives, linkers, and drugs. The drug delivery conjugatesdescribed herein are capable of selectively targeting a population ofpathogenic cells in the host animal due to preferential expression of areceptor for the binding ligand, such as a vitamin, accessible forligand binding, on the pathogenic cells. Illustrative vitamin moietiesthat can be used as the receptor binding ligand (B) include carnitine,inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenicacid, folic acid, riboflavin, thiamine, biotin, vitamin B₁₂, other watersoluble vitamins, the B vitamins, and the lipid soluble vitamins A, D, Eand K. These vitamins, and their receptor-binding analogs andderivatives, constitute an illustrative receptor binding ligand (B) thatcan be coupled with the therapeutic agent (A) drug by a linker (L) toform a drug delivery conjugate as described herein. The term vitamin isunderstood to include vitamin analogs and/or derivatives, unlessotherwise indicated. Illustratively, pteroic acid, which is a derivativeof folate, biotin analogs such as biocytin, biotin sulfoxide, oxybiotinand other biotin receptor-binding compounds, and the like, areconsidered to be vitamins, vitamin analogs, and vitamin derivatives. Itshould be appreciated that vitamin analogs or derivatives as describedherein refer to vitamins that incorporate an heteroatom through whichthe vitamin analog or derivative is covalently bound to the linker (L).

Illustrative vitamin moieties include folic acid, biotin, riboflavin,thiamine, vitamin B₁₂, and receptor-binding analogs and derivatives ofthese vitamin molecules, and other related vitamin receptor bindingmolecules.

In another embodiment, the cell receptor is a folate receptor, and thereceptor binding ligand (B) is a folate receptor binding ligand. Inanother embodiment, B is a folate, such as folic acid, or an analog orderivative of folic acid that binds to folic acid receptors. It is to beunderstood as used herein, that the term folate is used bothindividually and collectively to refer to folic acid itself, and/or tosuch analogs and derivatives of folic acid that are capable of bindingto folate receptors.

Illustrative embodiments of folate analogs and/or derivatives includefolinic acid, pteropolyglutamic acid, and folate receptor-bindingpteridines such as tetrahydropterins, dihydrofolates, tetrahydrofolates,and their deaza and dideaza analogs. The terms “deaza” and “dideaza”analogs refer to the art-recognized analogs having a carbon atomsubstituted for one or two nitrogen atoms in the naturally occurringfolic acid structure, or analog or derivative thereof. For example, thedeaza analogs include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and10-deaza analogs of folate. The dideaza analogs include, for example,1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs offolate. Other folates useful as complex forming ligands include thefolate receptor-binding analogs aminopterin, amethopterin(methotrexate), N¹⁰-methylfolate, 2-deamino-hydroxyfolate, deaza analogssuch as 1-deazamethopterin or 3-deazamethopterin, and3′,5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid(dichloromethotrexate). The foregoing folic acid analogs and/orderivatives are conventionally termed folates, reflecting their abilityto bind with folate-receptors, and such ligands when conjugated withexogenous molecules are effective to enhance transmembrane transport,such as via folate-mediated endocytosis as described herein.

Additional analogs of folic acid that bind to folic acid receptors aredescribed in U.S. Patent Application Publication Nos. 2005/0227985 and2004/0242582, the disclosures of which are incorporated herein byreference. Illustratively, such folate analogs have the general formula:

wherein X and Y are each-independently selected from the groupconsisting of halo, R², OR², SR³, and NR⁴R⁵;

U, V, and W represent divalent moieties each independently selected fromthe group consisting of —(R^(6a))C═, —N═, —(R^(6a))C(R^(7a))—, and—N(R^(4a))—; Q is selected from the group consisting of C and CH; T isselected from the group consisting of S, O, N, and —C═C—;

C¹ and C² are each independently selected from the group consisting ofoxygen, sulfur, —C(Z)—, —C(Z)O—, —OC(Z)—, —N(R^(4b))—, —C(Z)N(R^(4b))—,—N(R^(4b))C(Z)—, —OC(Z)N(R^(4b))—, —N(R^(4b))C(Z)O—,—N(R^(4b))C(Z)N(R^(5b))—, —S(O)—, —S(O)₂—, —N(R^(4a))S(O)₂—,—C(R^(6b))(R^(7b))—, —N(C≡CH)—, —N(CH₂C≡CH)—, C₁-C₁₂ alkylene, andC₁-C₁₂ alkyeneoxy, where Z is oxygen or sulfur;

R¹ is selected-from the group consisting of hydrogen, halo, C₁-C₁₂alkyl, and C₁-C₁₂ alkoxy; R², R³, R⁴, R^(4a), R^(4b), R⁵, R^(5b),R^(6b), and R^(7b) are each independently selected from the groupconsisting of hydrogen, halo, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₁-C₁₂alkanoyl, C₁-C₁₂ alkenyl, C₁-C₁₂ alkynyl, (C₁-C₁₂ alkoxy)carbonyl, and(C₁-C₁₂ alkylamino)carbonyl;

R⁶ and R⁷ are each independently selected from the group consisting ofhydrogen, halo, C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or, R⁶ and R⁷ are takentogether to form a carbonyl group; R^(6a) and R^(7a) are eachindependently selected from the group consisting of hydrogen, halo,C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or R^(6a) and R^(7a) are taken togetherto form a carbonyl group; and p, r, s, and t are each independentlyeither 0 or 1.

As used herein, it is to be understood that the term folate refers bothindividually to folic acid used in forming a drug delivery conjugate, oralternatively to a folate analog or derivative thereof that is capableof binding to folate receptors.

In one aspect of such folate analogs, when s is 1, t is 0, and when s is0, t is 1. In another aspect of such folate analogs, r is 1, and C² ofthe folate analog is covalently linked to a naturally occurring aminoacid at its alpha-amino group through an amide bond. Illustrative aminoacids include aspartic acid, glutamic acid, lysine, cysteine, and thelike.

The vitamin can be a folate which includes a nitrogen, and in thisembodiment, the spacer linkers can be alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹, and the spacer linker isbonded to the folate nitrogen to form an imide or an alkylamide.

In the various embodiments described herein, the substituents X¹ can bealkyl, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, carboxy, carboxyalkyl,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides.

Illustrative embodiments of vitamin analogs and/or derivatives alsoinclude analogs and derivatives of biotin such as biocytin, biotinsulfoxide, oxybiotin and other biotin receptor-binding compounds, andthe like. It is appreciated that analogs and derivatives of the othervitamins described herein are also contemplated herein. In oneembodiment, vitamins that can be used as the receptor binding ligand (B)in the drug delivery conjugates described herein include those that bindto vitamin receptors expressed specifically on activated macrophages oractivated monocytes, such as the folate receptor, which binds folate, oran analog or derivative thereof as described herein.

The linker L includes one or more hydrophilic spacer linkers.Illustrative hydrophilic linkers are described in WO2009/002993 and U.S.patent application Ser. No. 12/660,712, the disclosure of which isincorporated by reference herein in its entirety. In addition, otheroptional spacer linkers and/or releasable linkers may be included in L.It is appreciated that additional spacer linkers may be included whenpredetermined lengths are selected for separating receptor bindingligand (B) from therapeutic agent (A). It is also appreciated that incertain configurations, releasable linkers may be included. For example,as described herein in one embodiment, the drug delivery conjugates maybe used to deliver therapeutic agents (A) (e.g. drugs) for treatinginflammation. In such embodiments, it is appreciated that oncedelivered, the therapeutic agent (A) is desirably released from theconjugate. For example, in the configuration where the receptor bindingligand (B) is folate, or an analog or derivative thereof, the conjugatemay bind to a folate receptor. Once bound, the conjugate often undergoesthe process of endocytosis, and the conjugate is delivered to theinterior of the cell. Cellular mechanisms may biologically degrade theconjugate to release the drug “payload” and release the folate compound.

Accordingly, in other aspects, the conjugates B-L-A described hereinalso include the following general formulae:

B-L_(S)-L_(H)-A

B-L_(H)-L_(S)-A

B-L_(S)-L_(H)-L_(S)-A

B-L_(R)-L_(H)-A

B-L_(H)-L_(R)-A

B-L_(R)-L_(H)-L_(R)-A

B-L_(S)-L_(R)-L_(H)-A

B-L_(R)-L_(H)-L_(S)-A

B-L_(R)-L_(S)-L_(H)-L_(R)-A

B-L_(H)-L_(S)-L_(H)-L_(R)-A

where B, L, and A are as described herein, and L_(R) is a releasablelinker section, L_(S) is a spacer linker section, and L_(H) is ahydrophilic linker section of linker L. It is to be understood that theforegoing formulae are merely illustrative, and that other arrangementsof the hydrophilic spacer linker sections, releasable linker sections,and spacer linker sections are to be included herein. In addition, it isto be understood that additional conjugates are contemplated thatinclude a plurality hydrophilic spacer linkers, and/or a plurality ofreleasable linkers, and/or a plurality of spacer linkers.

It is appreciated that the arrangement and/or orientation of the varioushydrophilic linkers may be in a linear or branched fashion, or both. Forexample, the hydrophilic linkers may form the backbone of the linker (L)forming the conjugate between the folate and the drug (i.e. therapeuticagent (A)). Alternatively, the hydrophilic portion of the linker (L) maybe pendant to or attached to the backbone of the chain of atomsconnecting the receptor binding ligand B to the therapeutic agent A. Inthis latter arrangement, the hydrophilic portion may be proximal ordistal to the backbone chain of atoms.

In another embodiment, the linker (L) is more or less linear, and thehydrophilic groups are arranged largely in a series to form a chain-likelinker in the conjugate. Said another way, the hydrophilic groups formsome or all of the backbone of the linker (L) in this linear embodiment.

In another embodiment, the linker (L) is branched with hydrophilicgroups. In this branched embodiment, the hydrophilic groups may beproximal to the backbone or distal to the backbone. In each of thesearrangements, the linker (L) is more spherical or cylindrical in shape.In one variation, the linker (L) is shaped like a bottle-brush. In oneaspect, the backbone of the linker (L) is formed by a linear series ofamides, and the hydrophilic portion of the linker (L) is formed by aparallel arrangement of branching side chains, such as by connectingmonosaccharides, sulfonates, and the like, and derivatives and analogsthereof.

It is understood that the linker (L) may be neutral or ionizable undercertain conditions, such as physiological conditions encountered invivo. For ionizable linkers, under the selected conditions, the linker(L) may deprotonate to form a negative ion, or alternatively becomeprotonated to form a positive ion. It is appreciated that more than onedeprotonation or protonation event may occur. In addition, it isunderstood that the same linker (L) may deprotonate and protonate toform inner salts or zwitterionic compounds.

In another embodiment, the hydrophilic spacer linkers are neutral, i.e.under physiological conditions, the linkers do not significantlyprotonate nor deprotonate. In another embodiment, the hydrophilic spacerlinkers may be protonated to carry one or more positive charges. It isunderstood that the protonation capability is condition dependent. Inone aspect, the conditions are physiological conditions, and the linker(L) is protonated in vivo. In another embodiment, the hydrophilic spacerlinkers include both regions that are neutral and regions that may beprotonated to carry one or more positive charges. In another embodiment,the hydrophilic spacer linkers include both regions that may bedeprotonated to carry one or more negative charges and regions that maybe protonated to carry one or more positive charges. It is understoodthat in this latter embodiment that zwitterions or inner salts may beformed.

In one aspect, the regions of the linkers (L) that may be deprotonatedto carry a negative charge include carboxylic acids, such as asparticacid, glutamic acid, and longer chain carboxylic acid groups, andsulfuric acid esters, such as alkyl esters of sulfuric acid. In anotheraspect, the regions of the linkers (L) that may be protonated to carry apositive charge include amino groups, such as polyaminoalkylenesincluding ethylene diamines, propylene diamines, butylene diamines andthe like, and/or heterocycles including pyrollidines, piperidines,piperazines, and other amino groups, each of which is optionallysubstituted. In another embodiment, the regions of the hydrophilicspacer linkers that are neutral include poly hydroxyl groups, such assugars, carbohydrates, saccharides, inositols, and the like, and/orpolyether groups, such as polyoxyalkylene groups includingpolyoxyethylene, polyoxypropylene, and the like.

In one embodiment, the hydrophilic spacer linkers described herein areformed primarily from carbon, hydrogen, and oxygen, and have acarbon/oxygen ratio of about 3:1 or less, or of about 2:1 or less. Inone aspect, the hydrophilic linkers described herein include a pluralityof ether functional groups. In another aspect, the hydrophilic linkersdescribed herein include a plurality of hydroxyl functional groups.Illustrative fragments that may be used to form such linkers includepolyhydroxyl compounds such as carbohydrates, polyether compounds suchas polyethylene glycol (PEG) units, and acid groups such as carboxyl andalkyl sulfuric acids. In one variation, oligoamide spacers, and the likemay also be included in the linker (L).

Illustrative carbohydrate spacers include saccharopeptides as describedherein that include both a peptide feature and sugar feature;glucuronides, which may be incorporated via [2+3] Huisgen cyclization,also known as click chemistry; β-alkyl glycosides, such as of2-deoxyhexapyranoses (2-deoxyglucose, 2-deoxyglucuronide, and the like),and β-alkyl mannopyranosides.

In another illustrative embodiment, the hydrophilic spacer linkersdescribed herein include a plurality of hydroxyl functional groups, suchas linkers (L) that incorporate monosaccharides, oligosaccharides,polysaccharides, and the like. It is to be understood that thepolyhydroxyl containing spacer linkers comprises a plurality of —(CROH)—groups, where R is hydrogen or alkyl.

In another embodiment, the hydrophilic spacer linkers include one ormore of the following fragments:

wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an independentlyselected integer from 1 to about 3; n is an integer from 1 to about 6, pis an integer from 1 to about 5, and r is an integer selected from 1 toabout 3. In one variation, the integer n is 3 or 4. In anothervariation, the integer p is 3 or 4. In another variation, the integer ris 1.

In another embodiment, the hydrophilic spacer linkers described hereinare formed primarily from carbon, hydrogen, and nitrogen, and have acarbon/nitrogen ratio of about 3:1 or less, or of about 2:1 or less. Inone aspect, the hydrophilic linkers described herein include a pluralityof amino functional groups.

It is understood, that in such polyhydroxyl, polyamino, carboxylic acid,sulfuric acid, and like linkers that include free hydrogens bound toheteroatoms, one or more of those free hydrogen atoms may be protectedwith the appropriate hydroxyl, amino, or acid protecting group,respectively, or alternatively may be blocked as the correspondingpro-drugs, the latter of which are selected for the particular use, suchas pro-drugs that release the parent drug under general or specificphysiological conditions.

In each of the foregoing illustrative examples of linkers L, there arealso included in some cases additional spacer linkers Ls, and/oradditional releasable linkers L_(R). Those spacer linker and releasablelinkers also may include asymmetric carbon atoms. It is to be furtherunderstood that the stereochemical configurations shown herein aremerely illustrative, and other stereochemical configurations arecontemplated. It is to be further understood that in the foregoingembodiments, open positions, such as (*) atoms are locations forattachment of the receptor binding ligand (B) or the therapeutic agent(A) to be delivered. In addition, it is to be understood that suchattachment of either or both of B and A may be direct or through anintervening linker (L). Intervening linkers include other spacer linkersand/or releasable linkers. Illustrative additional spacer linkers andreleasable linkers that are included in the conjugate described hereinare described in U.S. Pat. No. 7,601,332, the disclosure of which isincorporated herein by reference.

In one embodiment, the hydrophilic spacer linker comprises one or morecarbohydrate containing or polyhydroxyl group containing linkers. Inanother embodiment, the hydrophilic spacer linker comprises at leastthree carbohydrate containing or polyhydroxyl group containing linkers.In another embodiment, the hydrophilic spacer linker comprises one ormore carbohydrate containing or polyhydroxyl group containing linkers,and one or more aspartic acids. In another embodiment, the hydrophilicspacer linker comprises one or more carbohydrate containing orpolyhydroxyl group containing linkers, and one or more glutamic acids.In another embodiment, the hydrophilic spacer linker comprises one ormore carbohydrate containing or polyhydroxyl group containing linkers,one or more glutamic acids, one or more aspartic acids, and one or morebeta amino alanines. In a series of variations, in each of the foregoingembodiments, the hydrophilic spacer linker also includes one or morecysteines. In another series of variations, in each of the foregoingembodiments, the hydrophilic spacer linker also includes at least onearginine.

In another series of variations, in each of the foregoing embodiments,the hydrophilic spacer linker also includes at least one arginine.

Illustrative spacer linkers include carbonyl, thionocarbonyl, alkylene,cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl,1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl,alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of saidspacer linkers is optionally substituted with one or more substituentsX¹ as defined herein.

Illustrative releasable linkers include methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,haloalkylenecarbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,(diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy,oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideniminyl,iminocycloalkylidenyl, carbonylcycloalkylideniminyl, alkylenethio,alkylenearylthio, and carbonylalkylthio, wherein each of the releasablelinkers is optionally substituted with a substituent X², as definedherein.

In any of the embodiments described herein, the substituents X² can bealkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl,alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,alkylthioalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl,alkyl carboxylate, alkyl alkanoate, guanidinoalkyl, R⁴-carbonyl,R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl, wherein R⁴ and R⁵are each independently selected from amino acids, amino acidderivatives, and peptides, and wherein R⁶ and R⁷ are each independentlyselected from amino acids, amino acid derivatives, and peptides. In thisembodiment the releasable linker can include nitrogen, and thesubstituent X² and the releasable linker can form an heterocycle.

In any of the embodiments described herein, the substituents X¹ can bealkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl,alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,alkylthioalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl,alkyl carboxylate, alkyl alkanoate, guanidinoalkyl, R⁴-carbonyl,R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl, wherein R⁴ and R⁵are each independently selected from the group consisting of an aminoacid, an amino acid derivative, and a peptide, and wherein R⁶ and R⁷ areeach independently selected from the group consisting of an amino acid,an amino acid derivative, and a peptide.

In any of the embodiments described herein, the substituents X¹ can bealkyl, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, carboxy, carboxyalkyl,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides.

The term cycloalkylene as used herein refers to a bivalent chain ofcarbon atoms, a portion of which forms a ring, such ascycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl,3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl, and the like.

The term aryl as used herein refers to an aromatic mono or polycyclicring of carbon atoms, such as phenyl, naphthyl, and the like. Inaddition, aryl may also include heteroaryl.

The term substituted aryl as used herein refers to the replacement ofone or more hydrogen atoms, generally on carbon, with a correspondingnumber of substituents, such as halo, hydroxy, amino, alkyl ordialkylamino, alkoxy, alkylsulfonyl, cyano, nitro, and the like.

The term heteroaryl as used herein refers to an aromatic mono orpolycyclic ring of carbon atoms and at least one heteroatom selectedfrom nitrogen, oxygen, and sulfur, such as pyridinyl, pyrimidinyl,indolyl, benzoxazolyl, and the like.

The term substituted heteroaryl as used herein refers to the replacementof one or more hydrogen atoms, generally on carbon, with a correspondingnumber of substituents, such as halo, hydroxy, amino, alkyl ordialkylamino, alkoxy, alkylsulfonyl, cyano, nitro, and the like.

The term optionally substituted as used herein refers to the replacementof one or more hydrogen atoms, generally on carbon, with a correspondingnumber of substituents, such as halo, hydroxy, amino, alkyl ordialkylamino, alkoxy, alkylsulfonyl, cyano, nitro, and the like. Inaddition, two hydrogens on the same carbon, on adjacent carbons, ornearby carbons may be replaced with a bivalent substituent to form thecorresponding cyclic structure.

The term amino acid as used herein refers generally toaminoalkylcarboxylate, where the alkyl radical is optionallysubstituted, such as with alkyl, hydroxy alkyl, sulfhydrylalkyl,aminoalkyl, carboxyalkyl, and the like, including groups correspondingto the naturally occurring amino acids, such as serine, cysteine,methionine, aspartic acid, glutamic acid, and the like. It is to beunderstood that such amino acids may be of a single stereochemistry or aparticular mixture of stereochemisties, including racemic mixtures. Inaddition, amino acid refers to beta, gamma, and longer amino acids, suchas amino acids of the formula:

—N(R)—(CR′R″)_(q)—C(O)—

where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and q is an integer such as1, 2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, ornithine, threonine, and thelike. In another illustrative aspect of the vitamin drug deliveryconjugate intermediate described herein, the drug (i.e., therapeuticagents (A)), or an analog or a derivative thereof, includes analkylthiol nucleophile.

As used herein the term “antifolate” refers to a compound thatinterferes with the metabolism of folic acid and its derivatives incellular processes.

It is to be understood that the above-described terms can be combined togenerate chemically-relevant groups, such as alkoxyalkyl referring tomethyloxymethyl, ethyloxyethyl, and the like, haloalkoxyalkyl referringto trifluoromethyloxyethyl, 1,2-difluoro-2-chloroeth-1-yloxypropyl, andthe like, arylalkyl referring to benzyl, phenethyl, α-methylbenzyl, andthe like, and others.

The term amino acid derivative as used herein refers generally to anoptionally substituted aminoalkylcarboxylate, where the amino groupand/or the carboxylate group are each optionally substituted, such aswith alkyl, carboxylalkyl, alkylamino, and the like, or optionallyprotected. In addition, the optionally substituted intervening divalentalkyl fragment may include additional groups, such as protecting groups,and the like.

The term peptide as used herein refers generally to a series of aminoacids and/or amino acid analogs and derivatives covalently linked one tothe other by amide bonds.

The term “releasable linker” as used herein refers to a linker (L) thatincludes at least one bond that can be broken under physiologicalconditions (e.g., a pH-labile, acid-labile, oxidatively-labile, orenzyme-labile bond). It should be appreciated that such physiologicalconditions resulting in bond breaking include standard chemicalhydrolysis reactions that occur, for example, at physiological pH, or asa result of compartmentalization into a cellular organelle such as anendosome having a lower pH than cytosolic pH.

The cleavable bond or bonds may be present in the interior of acleavable linker and/or at one or both ends of a cleavable linker. It isappreciated that the lability of the cleavable bond may be adjusted byincluding functional groups or fragments within the linker L that areable to assist or facilitate such bond breakage, also termed anchimericassistance. In addition, it is appreciated that additional functionalgroups or fragments may be included within the linker L that are able toassist or facilitate additional fragmentation of the conjugates afterbond breaking of the releasable linker. The lability of the cleavablebond can be adjusted by, for example, substitutional changes at or nearthe cleavable bond, such as including alpha branching adjacent to acleavable disulfide bond, increasing the hydrophobicity of substituentson silicon in a moiety having a silicon-oxygen bond that may behydrolyzed, homologating alkoxy groups that form part of a ketal oracetal that may be hydrolyzed, and the like.

It is understood that a cleavable bond can connect two adjacent atomswithin the releasable linker and/or connect other linkers (L) or Band/or A, as described herein, at either or both ends of the releasablelinker. In the case where a cleavable bond connects two adjacent atomswithin the releasable linker, following breakage of the bond, thereleasable linker is broken into two or more fragments. Alternatively,in the case where a cleavable bond is between the releasable linker andanother moiety, such as an additional heteroatom, additional spacerlinker, another releasable linker, the therapeutic agent A, or analog orderivative thereof, or the receptor binding ligand B, or analog orderivative thereof, following breakage of the bond, the releasablelinker is separated from the other moiety.

It is understood that each of the additional spacer and releasablelinkers are bivalent. It should be further understood that theconnectivity between each of the various additional spacer andreleasable linkers themselves, and between the various additional spacerand releasable linkers and A and/or B, as defined herein, may occur atany atom found in the various additional spacer or releasable linkers.

In another aspect, the linker (L) comprises a releasable linker, anadditional spacer linker, and a releasable linker taken together to formdithioalkylcarbonylhydrazide, where the hydrazide forms a hydrazide withthe therapeutic agent A, or analog or derivative thereof.

In another aspect, the linker (L) comprises a plurality of additionalspacer linkers selected from the group consisting of the naturallyoccurring amino acids and stereoisomers thereof.

In another aspect, the linker (L) comprises a releasable linker, anadditional spacer linker, and a releasable linker taken together to form2-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with theagent A, or analog or derivative thereof.

In another aspect, the linker (L) comprises a releasable linker, anadditional spacer linker, and a releasable linker taken together to form2-dithioalkyloxycarbonylhydrazide.

In another aspect, the linker (L) comprises a releasable linker, anadditional spacer linker, and a releasable linker taken together to form2-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe therapeutic agent A, or analog or derivative thereof.

In another aspect, the linker (L) comprises a releasable linker, anadditional spacer linker, and a releasable linker taken together to form2-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe therapeutic agent A, or analog or derivative thereof, and the alkylis ethyl.

In another aspect, the linker (L) comprises a releasable linker, anadditional spacer linker, and a releasable linker taken together to form2-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or acarbamoylaziridine with the therapeutic agent A, or analog or derivativethereof.

In one aspect, the releasable and spacer linkers may be arranged in sucha way that subsequent to the cleavage of a bond in the linker (L),released functional groups chemically assist the breakage or cleavage ofadditional bonds, also termed anchimeric assisted cleavage or breakage.

Illustrative mechanisms for cleavage of the linkers described hereininclude the following 1,4 and 1,6 fragmentation mechanisms

where X is an exogenous or endogenous nucleophile, glutathione, orbioreducing agent, and the like, and either of Z or Z′ is the vitamin(e.g. folate), or analog or derivative thereof, or the drug, or analogor derivative thereof, or a vitamin (e.g. folate) or drug in conjunctionwith other portions of the linker (L). It is to be understood thatalthough the above fragmentation mechanisms are depicted as concertedmechanisms, any number of discrete steps may take place to effect theultimate fragmentation of the linker (L) to the final products shown.For example, it is appreciated that the bond cleavage may also occur byacid-catalyzed elimination of the carbamate moiety, which may beanchimerically assisted by the stabilization provided by either the arylgroup of the beta sulfur or disulfide illustrated in the above examples.In those variations of this embodiment, the releasable linker is thecarbamate moiety. Alternatively, the fragmentation may be initiated by anucleophilic attack on the disulfide group, causing cleavage to form athiolate. The thiolate may intermolecularly displace a carbonic acid orcarbamic acid moiety and form the corresponding thiacyclopropane. In thecase of the benzyl-containing linkers, following an illustrativebreaking of the disulfide bond, the resulting phenyl thiolate mayfurther fragment to release a carbonic acid or carbamic acid moiety byforming a resonance stabilized intermediate. In any of these cases, thereleasable nature of the illustrative linkers described herein may berealized by whatever mechanism may be relevant to the chemical,metabolic, physiological, or biological conditions present.

It is to be understood that although the above fragmentation mechanismsare depicted as concerted mechanisms, any number of discrete steps maytake place to effect the ultimate fragmentation of the linker (L) to thefinal products shown. Alternatively, the fragmentation may be initiatedby a nucleophilic attack on the disulfide group, causing cleavage toform a thiolate. The thiolate may intermolecularly displace a carbonicacid or carbamic acid moiety and form the correspondingthiacyclopropane. In any of these cases, the releasable nature of theillustrative linkers (L) described herein may be realized by whatevermechanism may be relevant to the chemical, metabolic, physiological, orbiological conditions present. Without being bound by theory, in thisembodiment, acid catalysis, such as in an endosome, may also initiatethe cleavage via protonation of the urethane group. In addition,acid-catalyzed elimination of the carbamate leads to the release of CO₂and the nitrogen-containing moiety attached to Z, and the formation of abenzyl cation, which may be trapped by water, or any other Lewis base,as is similarly described herein.

In one embodiment, the linkers (L) described herein are compounds of thefollowing formulae

where n is an integer selected from 1 to about 4; R^(a) and R^(b) areeach independently selected from the group consisting of hydrogen andalkyl, including lower alkyl such as C₁-C₄ alkyl that are optionallybranched; or R^(a) and R^(b) are taken together with the attached carbonatom to form a carbocyclic ring; R is an optionally substituted alkylgroup, an optionally substituted acyl group, or a suitably selectednitrogen protecting group; and (*) indicates points of attachment forthe drug, folate, other linkers (L), or other parts of the conjugate.Another illustrative mechanism involves an arrangement of the releasableand spacer linkers in such a way that subsequent to the cleavage of abond in the linker (L), released functional groups chemically assist thebreakage or cleavage of additional bonds, also termed anchimericassisted cleavage or breakage.

In another illustrative embodiment, the linker (L) includes one or moreamino acids. In one variation, the linker (L) includes a single aminoacid. In another variation, the linker (L) includes a peptide havingfrom 2 to about 50, 2 to about 30, or 2 to about 20 amino acids. Inanother variation, the linker (L) includes a peptide having from about 4to about 8 amino acids. Such amino acids are illustratively selectedfrom the naturally occurring amino acids, or stereoisomers thereof. Theamino acid may also be any other amino acid, such as any amino acidhaving the general formula:

—N(R)—(CR′R″)_(q)—C(O)—

where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and q is an integer such as1, 2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, ornithine, threonine, and thelike. In one variation, the releasable linker includes at least 2 aminoacids selected from asparagine, aspartic acid, cysteine, glutamic acid,lysine, glutamine, arginine, serine, ornithine, and threonine. Inanother variation, the releasable linker includes between 2 and about 5amino acids selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, omithine, and threonine. Inanother variation, the releasable linker includes a tripeptide,tetrapeptide, pentapeptide, or hexapeptide consisting of amino acidsselected from aspartic acid, cysteine, glutamic acid, lysine, arginine,and ornithine, and combinations thereof.

In one illustrative embodiment of the invention, a method for treating apatient with an inflammatory disease, the method comprising the step ofadministering to the patient a composition comprising a drug deliveryconjugate of the formula

BL(A¹)(A²)_(m)

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

m is 0 or 1;

B is a folate;

L is a linker that comprises one or more hydrophilic spacer linkers;

A¹ is an antifolate; and

A² has the formula

wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug foming group, and C(O)R^(D), where R^(D) is in eachinstance independently selected from the group consisting of hydrogen,and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl,arylalkyl, heteroaryl, and heteroarylalkyl, each of which is optionallysubstituted is described.

In another embodiment, a pharmaceutical composition comprising a drugdelivery conjugate of the formula

BL(A¹)(A²)_(m)

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

m is 0 or 1;

B is a folate;

L is a linker that comprises one or more hydrophilic spacer linkers;

A¹ is an antifolate; and

A² has the formula

wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug forming group, and C(O)R^(D), where R^(D) is ineach instance independently selected from the group consisting ofhydrogen, and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted is described.

In one aspect, B, L, A¹, and A² in the conjugate BLA¹(A²)_(m) areconnected as shown in the following formula:

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein m is 1 and A¹ and A² are eachcovalently attached to linker L is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein L is a linker of the formula

wherein * indicates the point of attachment to the folate; ** indicatesthe point of attachment to one of A¹ or A²; *** indicates the point ofattachment to the remaining A¹ or A²; F and G are each independently 1,2, 3 or 4; and W¹ is NH or O is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the folate is of the formula

wherein * indicates the point of attachment to the linker;

X and Y are each-independently selected from the group consisting ofhalo, R², OR², SR³, and NR⁴R⁵;

U, V, and W represent divalent moieties each independently selected fromthe group consisting of —(R^(6a))C═, —N═, —(R^(6a))C(R^(7a))—, and—N(R^(4a))—; Q is selected from the group consisting of C and CH; T isselected from the group consisting of S, O, N, and —C═C—;

C¹ and C² are each independently selected from the group consisting ofoxygen, sulfur, —C(Z)—, —C(Z)O—, —OC(Z)—, —N(R^(4b))—, —C(Z)N(R^(4b))—,—N(R^(4b))C(Z)—, —OC(Z)N(R^(4b))—, —N(R^(4b))C(Z)O—,—N(R^(4b))C(Z)N(R^(5b))—, —S(O)—, —S(O)₂—, —N(R^(4a))S(O)₂—,—C(R^(6b))(R^(7b))—, —N(C≡CH)—, —N(CH₂C≡CH)—, C₁-C₁₂ alkylene, andC₁-C₁₂ alkyeneoxy, where Z is oxygen or sulfur;

R¹ is selected-from the group consisting of hydrogen, halo, C₁-C₁₂alkyl, and C₁-C₁₂ alkoxy; R², R³, R⁴, R^(4a), R^(4b), R⁵, R^(5b),R^(6b), and R^(7b) are each independently selected from the groupconsisting of hydrogen, halo, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₁-C₁₂alkanoyl, C₁-C₁₂ alkenyl, C₁-C₁₂ alkynyl, (C₁-C₁₂ alkoxy)carbonyl, and(C₁-C₁₂ alkylamino)carbonyl;

R⁶ and R⁷ are each independently selected from the group consisting ofhydrogen, halo, C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or, R⁶ and R⁷ are takentogether to form a carbonyl group; R^(6a) and R^(7a) are eachindependently selected from the group consisting of hydrogen, halo,C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or R^(6a) and R^(7a) are taken togetherto form a carbonyl group; and

p, r, s and t are each independently either 0 or 1 is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the antifolate is aminopterin,or an analog, or derivative, thereof is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the antifolate is aminopterinhydrazide is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the folate is of the formula

wherein * indicates the point of attachment to the linker is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein m¹ is 1; R^(A) and R^(B) arehydrogen; Y^(A) is OCH₂CH₂OR^(C); and R^(C) is a bond connected to L isdescribed.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein F is 2 and G is 1 is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the drug delivery conjugate isof the formula

is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the drug delivery conjugate isof the formula

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the drug delivery conjugate isof the formula

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the composition furthercomprises one or more carriers, diluents, or excipients, or acombination thereof is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the purity of the drug deliveryconjugate is at least 98% is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the composition is in a dosageform adapted for parenteral administration is described.

In another embodiment, the method of any one of the precedingembodiments wherein the dose of the drug delivery conjugate is in therange of 1 to 5 μg/kg is described.

In another embodiment, the method of any one of the precedingembodiments wherein the dose of the drug delivery conjugate is in therange of 1 to 3 μg/kg is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the disease is selected fromthe group consisting of arthritis, including rheumatoid arthritis andosteoarthritis, glomerulonephritis, proliferative retinopathy,restenosis, ulcerative colitis, Crohn's disease, fibromyalgia, psoriasisand other inflammations of the skin, inflammations of the eye, includinguveitis and autoimmune uveitis, osteomyelitis, Sjögren's syndrome,multiple sclerosis, diabetes, atherosclerosis, pulmonary fibrosis, lupuserythematosus, sarcoidosis, systemic sclerosis, organ transplantrejection (GVHD), and chronic inflammations is described.

In one embodiment, a compound having the following formula

is described.

In another embodiment, a kit comprising a sterile vial, the compositionor compound of any one of the preceding embodiments, and instructionsfor use describing use of the composition for treating a patient with aninflammatory disease is described.

In another embodiment, is described a kit comprising a sterile vial, acomposition comprising the compound as a lyophilized solid of any one ofthe preceding embodiments, and instructions describing use of thecomposition for treating a patient with an inflammatory disease, whereinthe vial is an amber glass vial with a rubber stopper and an aluminumtear-off seal.

In another embodiment, the kit of the preceding embodiment wherein thefolate is of the formula

wherein * indicates the point of attachment to the linker is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the antifolate is aminopterinhydrazide is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the antifolate is aminopterin hydrazide is described.

In another embodiment, the kit of any one of the preceding embodimentswherein in the conjugate m¹ is 1; R^(A) and R^(B) are hydrogen; Y^(A) isOCH₂CH₂OR^(C); and R^(C) is a bond connected to L is described.

In another embodiment, the kit of any one of the preceding embodimentswherein in the conjugate F is 2 and G is 1 is described.

In another embodiment, the kit of any one of the preceding embodimentsis described wherein the drug delivery conjugate is of the formula

In another embodiment, the kit of any one of the preceding embodimentsis described wherein the drug delivery conjugate is of the formula

In another embodiment, the kit of any one of the preceding embodimentswherein the drug delivery conjugate is of the formula

is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the composition or compound is in the form of a reconstitutablelyophlizate is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the dose of the drug delivery conjugate is in the range of 1 to5 μg/kg is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the dose of the drug delivery conjugate is in the range of 1 to3 μg/kg is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the purity of the drug delivery conjugate is at least 98% isdescribed.

In another embodiment, a kit is described wherein the compound of any ofthe preceding compound embodiments is lyophilized and is in the kitalong with a composition for reconstituting the lyophilizate.

In another embodiment, one of A is a derivative or analog of rapamycin.Illustrative examples of derivatives or analogs of rapamycin aredisclosed in U.S. Pat. Nos. 4,316,885, 4,650,803, 5,100,883, 5,118,677,5,118,678, 5,120,842, 5,130,307, 5,138,051, 5,151,413, 5,169,851,5,194,447, 5,221,670, 5,233,036, 5,258,389, 5,260,300, 5,302,584,5,362,718, 5,378,696, 5,385,908, 5,385,909, 5,385,910, 5,389,639,5,391,730, 5,463,048, and 5,491,231. The disclosure of each of theforegoing documents is incorporated by reference herein in its entirety.In another embodiment one of A is a derivative of everolimus.

In one illustrative embodiment of the invention a method for treating apatient with an inflammatory disease, the method comprising the step ofadministering to the patient a composition comprising a drug deliveryconjugate of the formula

B-L-A³

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

B is a folate;

L is a linker that comprises one or more hydrophilic spacer linkers; and

A³ has the formula

wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug forming group, and C(O)R^(D), where R^(D) is ineach instance independently selected from the group consisting ofhydrogen, and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted is described.

In another embodiment, a pharmaceutical composition comprising a drugdelivery conjugate of the formula

B-L-A³

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof;wherein

B is a folate;

L is a linker that comprises one or more hydrophilic spacer linkers; and

A³ has the formula

wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug forming group, and C(O)R^(D), where R^(D) is ineach instance independently selected from the group consisting ofhydrogen, and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, and C(O)R^(D), where R^(D) is in each instanceindependently selected from the group consisting of hydrogen, and alkyl,alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl,heteroaryl, and heteroarylalkyl, each of which is optionally substitutedis described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein L is a bivalent linker of theformula

wherein * indicates the point of attachment to the folate and **indicates the point of attachment to A³; and F and G are eachindependently 1, 2, 3 or 4 is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the folate is of the formula

wherein * indicates the point of attachment to the linker;

X and Y are each-independently selected from the group consisting ofhalo, R², OR², SR³, and NR⁴R⁵;

U, V, and W represent divalent moieties each independently selected fromthe group consisting of —(R^(6a))C═, —N═, —(R^(6a))C(R^(7a))—, and—N(R^(4a))—; Q is selected from the group consisting of C and CH; T isselected from the group consisting of S, O, N, and —C═C—;

A¹ and A² are each independently selected from the group consisting ofoxygen, sulfur, —C(Z)—, —C(Z)O—, —OC(Z)—, —N(R^(4b))—, —C(Z)N(R^(4b))—,—N(R^(4b))C(Z)—, —OC(Z)N(R^(4b))—, —N(R^(4b))C(Z)O—,—N(R^(4b))C(Z)N(R^(5b))—, —S(O)—, —S(O)₂—, —N(R^(4a))S(O)₂—,—C(R^(6b))(R^(7b))—, —N(C≡CH)—, —N(CH₂C≡CH)—, C₁-C₁₂ alkylene, andC₁-C₁₂ alkyeneoxy, where Z is oxygen or sulfur;

R¹ is selected-from the group consisting of hydrogen, halo, C₁-C₁₂alkyl, and C₁-C₁₂ alkoxy; R², R³, R⁴, R^(4a), R^(4b), R⁵, R^(5b),R^(6b), and R^(7b) are each independently selected from the groupconsisting of hydrogen, halo, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₁-C₁₂alkanoyl, C₁-C₁₂ alkenyl, C₁-C₁₂ alkynyl, (C₁-C₁₂ alkoxy)carbonyl, and(C₁-C₁₂ alkylamino)carbonyl;

R⁶ and R⁷ are each independently selected from the group consisting ofhydrogen, halo, C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or, R⁶ and R⁷ are takentogether to form a carbonyl group; R^(6a) and R^(7a) are eachindependently selected from the group consisting of hydrogen, halo,C₁-C₁₂ alkyl, and C₁-C₁₂ alkoxy; or R^(6a) and R^(7a) are taken togetherto form a carbonyl group; and

n, p, r, s and t are each independently either 0 or 1 is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the folate is of the formula

wherein * indicates the point of attachment to the linker is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein R^(A) and R^(B) are hydrogen;Y^(A) is OCH₂CH₂OR^(C); and R^(C) is a bond connected to L is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein F is 2 and G is 1 is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the drug delivery conjugate isof the formula

is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the composition furthercomprises one or more carriers, diluents, or excipients, or acombination thereof is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the purity of the drug deliveryconjugate is at least 98% is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the composition is in a dosageform adapted for parenteral administration is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the dose of the drug deliveryconjugate is in the range of 1 to 5 μg/kg is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the dose of the drug deliveryconjugate is in the range of 1 to 3 μg/kg is described.

In another embodiment, the method or pharmaceutical composition of anyone of the preceding embodiments wherein the disease is selected fromthe group consisting of arthritis, including rheumatoid arthritis andosteoarthritis, glomerulonephritis, proliferative retinopathy,restenosis, ulcerative colitis, Crohn's disease, fibromyalgia, psoriasisand other inflammations of the skin, osteomyelitis, Sjögren's syndrome,multiple sclerosis, diabetes, atherosclerosis, pulmonary fibrosis, lupuserythematosus, sarcoidosis, systemic sclerosis, organ transplantrejection (GVHD) and chronic inflammations is described.

In another embodiment, a kit comprising a sterile vial, the compositiondescribed in any of the preceding embodiments; and instructions for usedescribing use of the composition for treating a patient with aninflammatory disease is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the folate is of the formula

wherein * indicates the point of attachment to the linker is described.

In another embodiment, the kit of any one of the preceding embodimentswherein R^(A) and R^(B) are hydrogen; Y^(A) is OCH₂CH₂OR^(C); and R^(C)is a bond connected to L is described.

In another embodiment, the kit of any one of the preceding embodimentswherein F is 2 and G is 1 is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the drug delivery conjugate is of the formula

is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the composition is in the form of a reconstitutable lyophlizateis described.

In another embodiment, the kit of any one of the preceding embodimentswherein the dose of the drug delivery conjugate is in the range of 1 to5 μg/kg is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the dose of the drug delivery conjugate is in the range of 1 to3 μg/kg is described.

In another embodiment, the kit of any one of the preceding embodimentswherein the purity of the drug delivery conjugate is at least 98% isdescribed.

The conjugates or compounds described herein may contain one or morechiral centers, or may otherwise be capable of existing as multiplestereoisomers. It is to be understood that in one embodiment, theinvention described herein is not limited to any particularstereochemical requirement, and that the conjugates, compounds, andcompositions, methods, uses, and medicaments that include them may beoptically pure, or may be any of a variety of stereoisomeric mixtures,including racemic and other mixtures of enantiomers, other mixtures ofdiastereomers, and the like. It is also to be understood that suchmixtures of stereoisomers may include a single stereochemicalconfiguration at one or more chiral centers, while including mixtures ofstereochemical configuration at one or more other chiral centers.

Similarly, the compounds or conjugates described herein may be includegeometric centers, such as cis, trans, E, and Z double bonds. It is tobe understood that in another embodiment, the invention described hereinis not limited to any particular geometric isomer requirement, and thatthe conjugates, compounds, and compositions, methods, uses, andmedicaments that include them may be pure, or may be any of a variety ofgeometic isomer mixtures. It is also to be understood that such mixturesof geometric isomers may include a single configuration at one or moredouble bonds, while including mixtures of geometry at one or more otherdouble bonds.

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched. It is to be understood that alkyl isadvantageously of limited length, including C₁-C₂₄, C₁-C₁₂, C₁-C₈,C₁-C₆, and C₁-C₄. It is appreciated herein that shorter alkyl groups addless lipophilicity to the conjugate and accordingly will have differentpharmacokinetic behavior. As used herein, the term “cycloalkyl” includesa chain of carbon atoms, which is optionally branched, and where atleast a portion of the chain is cyclic. It is to be understood that achain forming cycloalkyl is advantageously of limited length, includingC₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₃-C₄. It is appreciated herein thatshorter alkyl groups add less lipophilicity to the conjugate andaccordingly will have different pharmacokinetic behavior.

As used herein, the term “heteroalkyl” includes a chain of atoms thatincludes both carbon and at least one heteroatom, and is optionallybranched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur.In certain variations, illustrative heteroatoms also include phosphorus,and selenium. As used herein, the term “heterocyclyl” includingheterocycle includes a chain of atoms that includes both carbon and atleast one heteroatom, and is optionally branched, where at least aportion of the chain is cyclic. Illustrative heteroatoms includenitrogen, oxygen, and sulfur. In certain variations, illustrativeheteroatoms also include phosphorus, and selenium. Illustrativeheteocycles include, but are not limited to, tetrahydrofuryl,pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl,homopiperazinyl, quinuclidinyl, and the like.

As used herein, the term “amino” includes the group NH₂, alkylamino, anddialkylamino, where the two alkyl groups in dialkylamino may be the sameor different, i.e. alkylalkylamino. Illustratively, amino includesmethylamino, ethylamino, dimethylamino, methylethylamino, and the like.In addition, it is to be understood that when amino modifies or ismodified by another term, such as aminoalkyl, or acylamino, the abovevariations of the term amino are included therein. Illustratively,aminoalkyl includes H₂N-alkyl, methylaminoalkyl, ethylaminoalkyl,dimethylaminoalkyl, methylethylaminoalkyl, and the like. Illustratively,acylamino includes acylmethylamino, acylethylamino, and the like.

As used herein, the term “optionally substituted amino” includesderivatives of amino as described herein, such as, but not limited to,acylamino, urea, and carbamate, and the like.

As used herein, the term “aryl” includes monocyclic and polycyclicaromatic carbocyclic and aromatic heterocyclic groups, each of which maybe optionally substituted. As used herein, the term “heteroaryl”includes aromatic heterocyclic groups, each of which may be optionallysubstituted. Illustrative carbocyclic aromatic groups described hereininclude, but are not limited to, phenyl, naphthyl, and the like.Illustrative heterocyclic aromatic groups include, but are not limitedto, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl,quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl,oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl,thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl,benzisoxazolyl, benzisothiazolyl, and the like.

The term “optionally substituted” as used herein includes thereplacement of hydrogen atoms with other functional groups on theradical that is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,nitro, sulfonic acids and derivatives thereof, carboxylic acids andderivatives thereof, and the like.

The term “optionally substituted aryl” as used herein includes thereplacement of hydrogen atoms with other functional groups on the arylthat is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,nitro, sulfonic acids and derivatives thereof, carboxylic acids andderivatives thereof, and the like.

Illustrative substituents include, but are not limited to, a radical—(CH₂)_(m)Z, where m is an integer from 0-6 and Z is selected fromhalogen, hydroxy, alkanoyloxy, including C₁-C₆ alkanoyloxy, optionallysubstituted aroyloxy, alkyl, including C₁-C₆ alkyl, alkoxy, includingC₁-C₆ alkoxy, cycloalkyl, including C₃-C₈ cycloalkyl, cycloalkoxy,including C₃-C₈ cycloalkoxy, alkenyl, including C₂-C₆ alkenyl, alkynyl,including C₂-C₆ alkynyl, haloalkyl, including C₁-C₆ haloalkyl,haloalkoxy, including C₁-C₆ haloalkoxy, halocycloalkyl, including C₃-C₈halocycloalkyl, halocycloalkoxy, including C₃-C₈ halocycloalkoxy, amino,C₁-C₆ alkylamino, (C₁-C₆ alkyl)(C₁-C₆ alkyl)amino, alkylcarbonylamino,N—(C₁-C₆ alkyl)alkylcarbonylamino, aminoalkyl, C₁-C₆ alkylaminoalkyl,(C₁-C₆ alkyl)(C₁-C₆ alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N—(C₁-C₆alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z is selected from—CO₂R⁴ and —CONR⁵R⁶, where R⁴, R⁵, and R⁶ are each independentlyselected in each occurrence from hydrogen, C₁-C₆ alkyl, and aryl-C₁-C₆alkyl.

The term “prodrug” as used herein generally refers to any conjugate thatwhen administered to a biological system generates a biologically activeconjugate as a result of one or more spontaneous chemical reaction(s),enzyme-catalyzed chemical reaction(s), and/or metabolic chemicalreaction(s), or a combination thereof. In vivo, the prodrug is typicallyacted upon by an enzyme (such as esterases, amidases, phosphatases, andthe like), simple biological chemistry, or other process in vivo toliberate or regenerate the more pharmacologically active drug. Thisactivation may occur through the action of an endogenous host enzyme ora non-endogenous enzyme that is administered to the host preceding,following, or during administration of the prodrug. Additional detailsof prodrug use are described in U.S. Pat. No. 5,627,165; and Pathalk etal., Enzymic protecting group techniques in organic synthesis,Stereosel. Biocatal. 775-797 (2000). It is appreciated that the prodrugis advantageously converted to the original drug as soon as the goal,such as targeted delivery, safety, stability, and the like is achieved,followed by the subsequent rapid elimination of the released remains ofthe group forming the prodrug.

Prodrugs may be prepared from the conjugate described herein byattaching groups, referred to as prodrug forming groups, that ultimatelycleave in vivo to one or more functional groups present on theconjugate, such as —OH—, —SH, —CO₂H, —NR₂. Illustrative prodrugs includebut are not limited to carboxylate esters where the group is alkyl,aryl, aralkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters ofhydroxyl, thiol and amines where the group attached is an acyl group, analkoxycarbonyl, aminocarbonyl, phosphate or sulfate. Illustrativeesters, also referred to as active esters, include but are not limitedto 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups such asacetoxymethyl, pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl,1-(cyclohexylcarbonyloxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl, andthe like; alkoxycarbonyloxyalkyl groups, such asethoxycarbonyloxymethyl, α-ethoxycarbonyloxyethyl,β-ethoxycarbonyloxyethyl, and the like; dialkylaminoalkyl groups,including di-lower alkylamino alkyl groups, such as dimethylaminomethyl,dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl, and the like;2-(alkoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like; and lactonegroups such as phthalidyl, dimethoxyphthalidyl, and the like.

Further illustrative prodrugs contain a chemical moiety, such as anamide or phosphorus group functioning to increase solubility and/orstability of the conjugates described herein. Further illustrativeprodrugs for amino groups include, but are not limited to,(C₃-C₂₀)alkanoyl; halo-(C₃-C₂₀)alkanoyl; (C₃-C₂₀)alkenoyl;(C₄-C₇)cycloalkanoyl; (C₃-C₆)-cycloalkyl(C₂-C₁₆)alkanoyl; optionallysubstituted aroyl, such as unsubstituted aroyl or aroyl substituted by 1to 3 substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with one or more of 1 to 3halogen atoms; optionally substituted aryl(C₂-C₁₆)alkanoyl, such as thearyl radical being unsubstituted or substituted by 1 to 3 substituentsselected from the group consisting of halogen, (C₁-C₃)alkyl and(C₁-C₃)alkoxy, each of which is optionally further substituted with 1 to3 halogen atoms; and optionally substituted heteroarylalkanoyl havingone to three heteroatoms selected from O, S and N in the heteroarylmoiety and 2 to 10 carbon atoms in the alkanoyl moiety, such as theheteroaryl radical being unsubstituted or substituted by 1 to 3substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl, and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with 1 to 3 halogen atoms. Thegroups illustrated are exemplary, not exhaustive, and may be prepared byconventional processes.

It is understood that the prodrugs themselves may not possesssignificant biological activity, but instead undergo one or morespontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s),and/or metabolic chemical reaction(s), or a combination thereof afteradministration in vivo to produce the conjugate described herein that isbiologically active or is a precursor of the biologically activeconjugate. However, it is appreciated that in some cases, the prodrug isbiologically active. It is also appreciated that prodrugs may oftenserve to improve drug efficacy or safety through improved oralbioavailability, pharmacodynamic half-life, and the like. Prodrugs alsorefer to derivatives of the conjugates described herein that includegroups that simply mask undesirable drug properties or improve drugdelivery. For example, one or more conjugates described herein mayexhibit an undesirable property that is advantageously blocked orminimized may become pharmacological, pharmaceutical, or pharmacokineticbarriers in clinical drug application, such as low oral drug absorption,lack of site specificity, chemical instability, toxicity, and poorpatient acceptance (bad taste, odor, pain at injection site, and thelike), and others. It is appreciated herein that a prodrug, or otherstrategy using reversible derivatives, can be useful in the optimizationof the clinical application of a drug.

It is appreciated that such hydrophilic linkers may alter the stability,metabolism and tissue distribution of the conjugates. For example, it isunderstood that in certain situations, carbohydrate-protein interactionsare weaker than peptide-protein interactions. Thus, it is appreciatedthat in various embodiments described herein, the conjugates may lead tolower binding of serum proteins. These and other physicochemicaldifferences between the conjugates described herein and others alreadyreported may include enhanced targeting to target cells and improved,i.e. more selective or differentially selective biodistributionprofiles. The increased cyctotoxicity may be a natural consequence ofthe decreased serum protein binding or the better or differentialbiodistribution (i.e. less drug is wasted in non-specific compartments).This is especially true for the use of hydrophilic but neutral spacers.Without being bound by theory it is also suggested that the hydrophilicspacer linkers described herein may decrease toxicity that might be dueat least in part to non-specific binding interactions.

In an alternate embodiment, the drug is linked to a hydrophilic spacerlinker, directly or indirectly, to accomplish the goal of decreasingliver clearance. It has been found herein that the attachment ofhydrophilic groups, either releasable or not, and more specificallyhydrophilic neutral groups, increases renal-specific delivery.

It has been observed that liver clearance of folate-drug conjugatespossessing disulfide linkers and peptidic spacers retain residual andsometimes substantial unfavorable toxicity profiles. Including thehydrophilic spacers described herein also introduced vectors forkidney-specific delivery. It is therefore appreciated that includingsuch linkers in the drug delivery conjugates may decrease overall liveruptake and consequentially decrease overall toxicity. Without beingbound by theory, it is appreciated that toxicity at MTD may be caused bynon-specific liver clearance, leading to metabolism and release of freedrug into bile and then the intestine. The local toxicity as well assystemic toxicity (due to re-absorption) might then occur. By includinghydrophilic linkers in the conjugates described herein, it is believedthat clearance through the kidney may preferentially occur, thusdecreasing and/or avoiding concomitant liver metabolism based toxicity.Accordingly, measuring total bile clearance of the drug component from aseries of drug-folate conjugates, may be used to predict which agentwould be the least toxic.

As described above, the conjugates described herein may be used todeliver therapeutic agents A (e.g. drugs) to cells in a selective orspecific manner. In one aspect of such delivery, unwanted clearancemechanisms may also be avoided. It has been discovered that thehydrophilic spacer linkers described herein when used to form conjugatesof receptor binding ligands B and therapeutic agents A, can decrease theamount of clearance by the liver. It has further been discovered thatthese hydrophilic spacer linkers tend to favor clearance along renalpathways, such as the kidney. It has further been discovered that theconjugates described herein exhibit lower toxicity than the parenttherapeutic agents A by themselves when administered in the same way.Without being bound by theory, it is suggested that the lower toxicityarises from the observed decrease in liver clearance mechanism in favorof renal clearance mechanisms.

In another embodiment, multi-drug conjugates are described herein.Several illustrative configurations of such multi-drug conjugates arecontemplated herein, and include the compounds and configurationsdescribed in PCT international publication No. WO 2007/022494, thedisclosure of which is incorporated herein by reference. In one aspect,the linker (L) can be a polyvalent linker. Illustratively, thepolyvalent linkers may connect the folate receptor binding ligand B tothe two or more therapeutic agents A (e.g. drug) in a variety ofstructural configurations

In one illustrative embodiment, one of the therapeutic agents (e.g.drugs) is aminopterin or aminopterin hydrazide. If an additional drug isincluded in the conjugate, it can be a drug of formula I or a differentdrug.

wherein

Y^(A) is OR^(C) or OCH₂CH₂OR^(C);

one of R^(A), R^(B), or R^(C) is a bond connected to L; and

the other two of R^(A), R^(B), and R^(C) are independently selected ineach case from the group consisting of hydrogen, optionally substitutedheteroalkyl, prodrug forming group, and C(O)R^(D), where R^(D) is ineach instance independently selected from the group consisting ofhydrogen, and alkyl, alkenyl, heteroalkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted. If the second drug is a drug different thanformula I, the second drug can be selected based on activity against oneor more populations of pathogenic cells, such as inflammatory cells,with a particular mechanism of action. Illustrative mechanisms of actioninclude alkylating agents, microtubule inhibitors, including those thatstabilize and/or destabilize microtubule formation, includingbeta-tubulin agents, cyclin dependent kinase (CDK) inhibitors,topoisomerase inhibitors, protein synthesis inhibitors, protein kinaseinhibitors, including inhibitors of Ras, Raf, PKC, PI3K, and likeinhibitors, transcription inhibitor, antifolates, heat shock proteinblockers, and the like.

Illustrative alkylating agents include, but are not limited to,mitomycins CBI, and the like. Illustrative cyclin dependent kinase (CDK)inhibitors include, but are not limited to, CYC202, seliciclib,R-roscovitine, AGM-1470, and the like. Illustrative topoisomeraseinhibitors include, but are not limited to, doxorubicin, otheranthracyclines, and the like. Illustrative protein synthesis inhibitorsinclude, but are not limited to, bruceantin, and the like. Illustrativeprotein kinase inhibitors, including inhibitors of Ras, Raf, PKC, PI3K,and like inhibitors, include but are not limited to L-779,450, R115777,and the like. Illustrative transcription inhibitors include, but are notlimited to, α-amanatin, actinomycin, and the like. Illustrativeantifolates include, but are not limited to, methotrexate, aminopterin,and the like. Illustrative heat shock protein blockers include, but arenot limited to, geldanamycin, and the like.

Illustrative microtubule inhibitors, including those that stabilizeand/or destabilize microtubule formation, include β-tubulin agents,microtubule poisons, and the like. Illustrative microtubule poisons thatbind to selected receptors include, but are not limited to, inhibitorsbinding to the vinca binding site such as arenastatin, dolastatin,halichondrin B, maytansine, phomopsin A, rhizoxin, ustiloxin,vinblastine, vincristine, and the like, stabilizers binding to the taxolbinding site such as discodermalide, epothilone, taxol, paclitaxol, andthe like, inhibitors binding to the colchicine binding site such as,colchicine, combretastatin, curacin A, podophyllotoxin, steganacine, andthe like, and others binding to undefined sites such as cryptophycin,tubulysins, and the like.

In one embodiment, the tubulsyin is a naturally occurring tubulysin. Inanother embodiment, the tubulsyin is a synthetic or semi-synthetictubulysin. Additional tubulysins that may be included in the conjugatesdescribed herein are described in PCT international application serialNo. PCT/US2008/056824, the disclosure of which is incorporated herein byreference.

In one aspect of the drug delivery conjugates described herein, at leastone of the drugs is an antifolate. In one illustrative example, theantifolate is aminopterin. In another illustrative example, theantifolate is aminopterin hydrazide. In other embodiments, where asecond drug is included, the second drug can be a DNA alkylation agent.In another embodiment, the second drug can be a microtubule inhibitor.

In another embodiment of the drug delivery conjugates described herein,the second drug is a P-glycoprotein (PGP) inhibitor.

In another embodiment of the drug delivery conjugates described herein,the second drug is a drug having formula I.

In another embodiment of the drug delivery conjugates described herein,the second drug is a vinca alkaloid, or an analog or derivative thereof.Vinca alklaloids described herein include all members of the vincaindole-dihydroindole family of alkaloids, such as but not limited tovindesine, vinblastine, vincristine, catharanthine, vindoline,leurosine, vinorelbine, vinblastinoic acid, and the like, and analogsand derivatives thereof.

In another embodiment, methods for treating diseases caused by orevidenced by pathogenic cell populations, such as inflammatory cells,are described herein. The drug delivery conjugates can be used to treatdisease states characterized by the presence of a pathogenic cellpopulation, such as inflammatory cells, in the host wherein the membersof the pathogenic cell population have an accessible binding site forthe folate, or analog or derivative thereof, wherein the binding site isuniquely expressed, overexpressed, or preferentially expressed by thepathogenic cells. The selective elimination of the pathogenic cells ismediated by the binding of the drug delivery conjugate to a receptor(e.g., a folate receptor when the conjugate is folate targeted), whichis uniquely expressed, overexpressed, or preferentially expressed by thepathogenic cells. A receptor (e.g., a folate receptor) uniquelyexpressed, overexpressed, or preferentially expressed by the pathogeniccells is not present or present at lower concentrations onnon-pathogenic cells providing a means for selective elimination of thepathogenic cells.

For example, surface-expressed vitamin receptors, such as thehigh-affinity folate receptor, are overexpressed activated macrophagesand activated monocytes. Accordingly, the drug delivery conjugatesdescribed herein can be used to treat a variety of inflammatory celltypes that preferentially express folate receptors, and, thus, havesurface accessible binding sites for ligands, such as folate, or folateanalogs or derivatives. In one aspect, methods are described herein fortargeting the conjugates to maximize targeting of the pathogenic cellsfor elimination.

The invention further contemplates the use of combinations of drugdelivery conjugates to maximize targeting of the pathogenic cells, suchas inflammatory cells, for elimination. In accordance with the invention“elimination”, “eliminated”, and “eliminating” a population of cellsmean completely eliminating a population of cells, eliminating somecells, or reducing the symptoms of disease caused by the cells, such asinflammatory cells.

The drug delivery conjugates described herein can be used for both humanclinical medicine and veterinary applications. Thus, the host animalharboring the population of pathogenic cells and treated with the drugdelivery conjugates (e.g., a folate conjugate) can be human or, in thecase of veterinary applications, can be a laboratory, agricultural,domestic, or wild animal. The methods described herein can be applied tohost animals including, but not limited to, humans, laboratory animalssuch rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys,chimpanzees, domestic animals such as dogs, cats, and rabbits,agricultural animals such as cows, horses, pigs, sheep, goats, and wildanimals in captivity such as bears, pandas, lions, tigers, leopards,elephants, zebras, giraffes, gorillas, dolphins, and whales.

The methods are applicable to populations of pathogenic cells that causeinflammation. For example, activated macrophages or activated monocytescapable of causing a disease state, such as inflammation, can beeliminated because they uniquely express, preferentially express, oroverexpress folate receptors, or receptors that bind analogs orderivatives of folate. For example, the pathogenic cells can beinflammatory cells that are pathogenic under some circumstances such ascells of the immune system that are responsible for graft versus hostdisease, but not pathogenic under other circumstances.

In one embodiment, the drug delivery conjugates can be internalized intothe targeted pathogenic cells upon binding of the binding ligand moiety(e.g. folate) to a receptor, transporter, or other surface-presentedprotein that specifically binds the ligand and which is preferentiallyexpressed on the pathogenic cells. Such internalization can occur, forexample, through receptor-mediated endocytosis. If the drug deliveryconjugate contains a releasable linker, the B moiety and the drug candissociate intracellularly and the drug can act on its intracellulartarget.

In an alternate embodiment, the B (e.g. folate) can bind to thepathogenic cell placing the drug in close association with the surfaceof the pathogenic cell. The drug can then be released by cleavage of thereleasable linker. For example, the drug can be released by a proteindisulfide isomerase if the releasable linker is a disulfide group. Thedrug can then be taken up by the pathogenic cell to which the drugdelivery conjugate is bound, or the drug can be taken up by anotherpathogenic cell in close proximity thereto. Alternatively, the drugcould be released by a protein disulfide isomerase inside the cell wherethe releasable linker is a disulfide group. The drug may also bereleased by a hydrolytic mechanism, such as acid-catalyzed hydrolysis,as described above for certain beta elimination mechanisms, or by ananchimerically assisted cleavage through an oxonium ion or lactonium ionproducing mechanism. The selection of the releasable linker or linkerswill dictate the mechanism by which the drug is released from theconjugate. It is appreciated that such a selection can be pre-defined bythe conditions wherein the drug conjugate will be used. Alternatively,the drug delivery conjugates can be internalized into the targeted cellsupon binding, and the receptor binding ligand (B) and the drug canremain associated intracellularly with the drug exhibiting its effectswithout dissociation from the vitamin moiety.

In still another embodiment where the receptor binding ligand (B) is afolate, the drug delivery conjugate can act through a mechanismindependent of cellular folate receptors. For example, the drug deliveryconjugates can bind to soluble folate receptors present in the serum orto serum proteins, such as albumin, resulting in prolonged circulationof the conjugates relative to the unconjugated drug, and in increasedactivity of the conjugates towards the pathogenic cell population, suchas inflammatory cells, relative to the unconjugated drug.

In another embodiment, where the linker (L) does not comprise areleasable linker, and B is folate, the folate moiety of the drugdelivery conjugate can bind to the pathogenic cell placing the drug onthe surface of the pathogenic cell to target the pathogenic cell forattack by other molecules capable of binding to the drug. Alternatively,in this embodiment, the drug delivery conjugates can be internalizedinto the targeted cells upon binding, and the vitamin moiety and thedrug can remain associated intracellularly with the drug exhibiting itseffects without dissociation from the folate.

The drug delivery conjugates described herein can comprise a receptorbinding ligand (B) (e.g. a folate), a linker (L), a drug, and,optionally, heteroatom linkers to link the receptor binding ligand (B)and the drug to the linker (L). The linker (L) can comprise a spacerlinker, a releasable (i.e., cleavable) linker, and an heteroatom linker,or combinations thereof.

In one embodiment, the drug is aminopterin. In another embodiment, asecond drug may be present. Suitable second drugs can include, but arenot limited to: peptides, oligopeptides, retro-inverso oligopeptides,proteins, protein analogs in which at least one non-peptide linkagereplaces a peptide linkage, apoproteins, glycoproteins, enzymes,coenzymes, enzyme inhibitors, amino acids and their derivatives,receptors and other membrane proteins; antigens and antibodies thereto;haptens and antibodies thereto; hormones, lipids, phospholipids,liposomes; toxins; analgesics; bronchodilators; beta-blockers;antihypertensive agents; cardiovascular agents includingantiarrhythmics, cardiac glycosides, antianginals and vasodilators;central nervous system agents including stimulants, psychotropics,antimanics, and depressants; antihistamines; tranquilizers;anti-depressants; H-2 antagonists; anticonvulsants; antinauseants;prostaglandins and prostaglandin analogs; muscle relaxants;anti-inflammatory substances; stimulants; decongestants; antiemetics;diuretics; antispasmodics; antiasthmatics; cough suppressants;mucolytics; mineral and nutritional additives; adrenocorticoids andcorticosteroids; alkylating agents; antiandrogens; antiestrogens;androgens; aclamycin and aclamycin derivatives; estrogens;antimetabolites such as cytosine arabinoside; purine analogs; pyrimidineanalogs; and methotrexate; busulfan; carboplatin; chlorambucil;cisplatin and other platinum compounds; tamoxiphen; taxol; paclitaxel;paclitaxel derivatives; Taxotere®; cyclophosphamide; daunomycin;daunorubicin; doxorubicin; rhizoxin; T2 toxin; plant alkaloids;prednisone; hydroxyurea; teniposide; mitomycins; discodermolides;microtubule inhibitors; epothilones; tubulysin; cyclopropylbenz[e]indolone; seco-cyclopropyl benz[e]indolone; O-Ac-seco-cyclopropylbenz[e]indolone; bleomycin and any other antibiotic; nitrogen mustards;nitrosureas; vincristine; vinblastine; analogs and derivative thereofsuch as deacetylvinblastine monohydrazide; and other vinca alkaloids;including those described in PCT international publication No. WO2007/022493; the disclosure of which is incorporated herein byreference; colchicine; colchicine derivatives; allocolchicine;thiocolchicine; trityl cysteine; Halicondrin B; dolastatins such asdolastatin 10; amanitins such as α-amanitin; camptothecin; irinotecan;and other camptothecin derivatives thereof; maytansines; geldanamycinand geldanamycin derivatives; estramustine; nocodazole; MAP4; colcemid;inflammatory and proinflammatory agents; peptide and peptidomimeticsignal transduction inhibitors; and any other art-recognized drug ortoxin.

In another embodiment, the second drug can be selected from a vincaalkaloid, such as DAVLBH, a cryptophycin, bortezomib, thiobortezomib, atubulysin, aminopterin, rapamycin, paclitaxel, docetaxel, doxorubicin,daunorubicin, everolimus, α-amanatin, verucarin, didemnin B,geldanomycin, purvalanol A, ispinesib, budesonide, dasatinib, anepothilone, a maytansine, and a tyrosine kinase inhibitor, includinganalogs and derivatives of the foregoing. In one variation, thetherapeutic agents (A) (e.g. drugs) are the same and are antifolatecompounds. In one variation, the therapeutic agents (A) (e.g. drugs) arethe same and are aminopterin hydrazide. In another variation, thetherapeutic agents (A) (e.g. drugs) are different, but at least one ofthe therapeutic agents (A) is an antifolate.

In one embodiment, the drugs for use in the methods described hereinremain stable in serum for at least 4 hours. In another embodiment thedrugs have an IC₅₀ in the nanomolar range, and, in another embodiment,the drugs are water soluble. If the drug is not water soluble, thelinker (L) can be derivatized to enhance water solubility. The term“drug” also means any of the drug analogs or derivatives describedhereinabove. It should be appreciated that in accordance with thisinvention, a drug analog or derivative can mean a drug that incorporatesa heteroatom through which the drug analog or derivative is covalentlybound to the linker (L).

The drug delivery conjugates can comprise a receptor binding ligand (B)(e.g. a folate), a linker (L), a drug, and, optionally, heteroatomlinkers to link the receptor binding ligand (B) and the drug to thelinker (L). In one illustrative embodiment, it should be appreciatedthat a folate analog or derivative can mean a folate that incorporates aheteroatom through which the folate analog or derivative is covalentlybound to the linker (L). Thus, in this illustrative embodiment, thefolate can be covalently bound to the linker (L) through a heteroatomlinker, or a vitamin analog or derivative (i.e., incorporating anheteroatom) can be directly bound to the linker (L). In similarillustrative embodiments, a drug analog or derivative is a drug, and adrug analog or derivative can mean a drug that incorporates anheteroatom through which the drug analog or derivative is covalentlybound to the linker (L). Thus, in these illustrative aspects, the drugcan be covalently bound to the linker (L) through an heteroatom linker,or a drug analog or derivative (i.e., incorporating an heteroatom) canbe directly bound to the linker (L). The linker (L) can comprise aspacer linker, a releasable (i.e., cleavable) linker, and a heteroatomlinker to link the spacer linker to the releasable linker in conjugatescontaining both of these types of linkers. The linker can be a bivalentlinker.

Generally, any manner of forming a conjugate between the linker (L) andthe folate or analog or derivative thereof, between the linker (L) andthe drug, or analog or derivative thereof, including any interveningheteroatom linkers, can be utilized. Also, any art-recognized method offorming a conjugate between the spacer linker, the releasable linker,and the heteroatom linker to form the bivalent linker can be used. Theconjugate can be formed by direct conjugation of any of these molecules,for example, through complexation, or through hydrogen, ionic, orcovalent bonds. Covalent bonding can occur, for example, through theformation of amide, ester, disulfide, or imino bonds between acid,aldehyde, hydroxy, amino, sulfhydryl, or hydrazo groups.

In another embodiment, the (L) linker includes a chain of atoms selectedfrom C, N, O, S, Si, and P that covalently connects the receptor bindingligand (B), the hydrophilic linker, and/or the therapeutic agent (A).The linker (L) may have a wide variety of lengths, such as in the rangefrom about 2 to about 100 atoms. The atoms used in forming the linker(L) may be combined in all chemically relevant ways, such as chains ofcarbon atoms forming alkylene, alkenylene, and alkynylene groups, andthe like; chains of carbon and oxygen atoms forming ethers,polyoxyalkylene groups, or when combined with carbonyl groups formingesters and carbonates, and the like; chains of carbon and nitrogen atomsforming amines, imines, polyamines, hydrazines, hydrazones, or whencombined with carbonyl groups forming amides, ureas, semicarbazides,carbazides, and the like; chains of carbon, nitrogen, and oxygen atomsforming alkoxyamines, alkoxylamines, or when combined with carbonylgroups forming urethanes, amino acids, acyloxylamines, hydroxamic acids,and the like; and many others. In addition, it is to be understood thatthe atoms forming the chain in each of the foregoing illustrativeembodiments may be either saturated or unsaturated, such that forexample, alkanes, alkenes, alkynes, imines, and the like may be radicalsthat are included in the linker (L). In addition, it is to be understoodthat the atoms forming the linker (L) may also be cyclized upon eachother to form divalent cyclic structures that form the linker, includingcyclo alkanes, cyclic ethers, cyclic amines, arylenes, heteroarylenes,and the like in the linker (L). The linker (L) may be bivalent.

In another embodiment, pharmaceutical compositions comprising an amountof a drug delivery conjugate effective to eliminate a population ofpathogenic cells, such as inflammatory cells, in a host animal whenadministered in one or more doses are described. The drug deliveryconjugate is preferably administered to the host animal parenterally,e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally,intravenously, or intrathecally. Alternatively, the drug deliveryconjugate can be administered to the host animal by other medicallyuseful processes, such as orally, and any effective dose and suitabletherapeutic dosage form, including prolonged release dosage forms, canbe used.

Examples of parenteral dosage forms include aqueous solutions of theconjugates, in an isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as liquid alcohols,glycols, esters, and amides. The parenteral dosage form can be in theform of a reconstitutable lyophilizate comprising the dose of the drugdelivery conjugate. In one aspect of the present embodiment, any of anumber of prolonged release dosage forms known in the art can beadministered such as, for example, the biodegradable carbohydratematrices described in U.S. Pat. Nos. 4,713,249; 5,266,333; and5,417,982, the disclosures of which are incorporated herein byreference, or, alternatively, a slow pump (e.g., an osmotic pump) can beused.

In one illustrative aspect, at least one additional compositioncomprising a therapeutic factor can be administered to the host incombination or as an adjuvant to the above-detailed methodology, toenhance the drug delivery conjugate-mediated elimination of thepopulation of pathogenic cells, or more than one additional therapeuticfactor can be administered. The therapeutic factor can be selected froma chemotherapeutic agent, or another therapeutic factor capable ofcomplementing the efficacy of the administered drug delivery conjugate.

In one illustrative aspect, therapeutically effective combinations ofthese factors can be used. In one embodiment, for example,therapeutically effective amounts of the therapeutic factor, forexample, in amounts ranging from about 0.1 MIU/m²/dose/day to about 15MIU/m²/dose/day in a multiple dose daily regimen, or for example, inamounts ranging from about 0.1 MIU/m²/dose/day to about 7.5MIU/m²/dose/day in a multiple dose daily regimen, can be used along withthe drug delivery conjugates to eliminate, reduce, or neutralizepathogenic cells in a host animal harboring the pathogenic cells(MIU=million international units; m²=approximate body surface area of anaverage human).

Additionally, more than one type of drug delivery conjugate can be used.Illustratively, for example, the patient can be treated with conjugateswith different vitamins (e.g. folates), but the same drug in a co-dosingprotocol. In other embodiments, the patient can be treated withconjugates comprising the same receptor binding ligand (B) (e.g. afolate) linked to different drugs, or various receptor binding ligands(B) linked to various drugs. In another illustrative embodiment, drugdelivery conjugates with the same or different vitamins (e.g. folates),and the same or different drugs comprising multiple vitamins andmultiple drugs as part of the same drug delivery conjugate could beused.

The unitary daily dosage of the drug delivery conjugate can varysignificantly depending on the host condition, the specific inflammatorydisease state being treated, the molecular weight of the conjugate, itsroute of administration and tissue distribution, and the possibility ofco-usage of other therapeutic treatments such as radiation therapy. Theeffective amount to be administered to a patient is based on bodysurface area, patient weight, and physician assessment of patientcondition. In illustrative embodiments, effective doses can range, forexample, from about 1 ng/kg to about 10 mg/kg, from about 100 ng toabout 1 mg, from about 1 μg/kg to about 500 μg/kg, from about 1 μg/kg toabout 100 μg/kg, from about 1 μg/kg to about 50 μg/kg, and from about 1μg/kg to about 10 μg/kg. The reference to kg is kg of patient bodyweight.

In another illustrative aspect, any effective regimen for administeringthe drug delivery conjugates can be used. For example, the drug deliveryconjugates can be administered as single doses, or can be divided andadministered as a multiple-dose daily regimen. In other embodiments, astaggered regimen, for example, one to three days per week can be usedas an alternative to daily treatment, and such intermittent or staggereddaily regimen is considered to be equivalent to every day treatment andwithin the scope of the methods described herein. In one embodiment, thepatient is treated with multiple injections of the drug deliveryconjugate to eliminate the population of pathogenic cells, such asinflammatory cells. In another embodiment, the patient is injectedmultiple times (preferably about 2 up to about 50 times) with the drugdelivery conjugate, for example, at 12-72 hour intervals or at 48-72hour intervals. In other embodiments, additional injections of the drugdelivery conjugate can be administered to the patient at an interval ofdays or months after the initial injections(s) and the additionalinjections prevent recurrence of the disease state caused by thepathogenic cells, such as inflammatory cells.

In one embodiment, folates, or analogs or derivatives thereof that canbe used in the drug delivery conjugates include those that bind tofolate receptors expressed specifically on activated macrophages oractivated monocytes. The folate-linked conjugates, for example, can beused to kill or suppress the activity of activated macrophages oractivated monocytes that cause disease states in the patient. Suchconjugates, when administered to a patient suffering from inflammation,work to concentrate and associate the conjugated drug in the populationof inflammatory cells to kill the inflammatory cells or suppress theirfunction. Elimination, reduction, or deactivation of the inflammatorycell population works to stop or reduce the pathogenesis characteristicof the disease state being treated. Exemplary of inflammatory diseasesinclude arthritis, including rheumatoid arthritis and osteoarthritis,glomerulonephritis, proliferative retinopathy, restenosis, ulcerativecolitis, Crohn's disease, fibromyalgia, psoriasis and otherinflammations of the skin, inflammations of the eye, including uveitisand autoimmune uveitis, osteomyelitis, Sjögren's syndrome, multiplesclerosis, diabetes, atherosclerosis, pulmonary fibrosis, lupuserythematosus, sarcoidosis, systemic sclerosis, organ transplantrejection (GVHD) and chronic inflammations is described Administrationof the drug delivery conjugate is typically continued until symptoms ofthe disease state are reduced or eliminated.

As used herein the term uveitis generally refers to an intraocularinflammatory disease including iritis, cyclitis, panuveits, posterioruveitis and anterior uveitis. Iritis is inflammation of the iris.Cyclitis is inflammation of the ciliary body. Panuveitis refers toinflammation of the entire uveal (vascular) layer of the eye.Intermediate uveitis, also called peripheral uveitis, is centered in thearea immediately behind the iris and lens in the region of the ciliarybody and pars plana, and is also termed “cyclitis” and “pars planitis.”

Autoimmune uveitis may occur as a component of an autoimmune disorder(such as rheumatoid arthritis, Bechet's disease, ankylosing spondylitis,sarcoidosis, and the like), as an isolated immune mediated oculardisorder (such as pars planitis or iridocyclitis, and the like), as adisease unassociated with known etiologies, and following certainsystemic diseases which cause antibody-antigen complexes to be depositedin the uveal tissues.

Illustratively, the drug delivery conjugates administered to killinflammatory cells or suppress their function can be administeredparenterally to the animal or patient suffering from the disease state,for example, intradermally, subcutaneously, intramuscularly,intraperitoneally, or intravenously in combination with apharmaceutically acceptable carrier. In another embodiment, the drugdelivery conjugates can be administered to the animal or patient byother medically useful procedures and effective doses can beadministered in standard or prolonged release dosage forms. In anotheraspect, the therapeutic method can be used alone or in combination withother therapeutic methods recognized for treatment of inflammation.

In other embodiments of the methods described herein, pharmaceuticallyacceptable salts of the conjugates described herein can be used.Pharmaceutically acceptable salts of the conjugates described hereininclude the acid addition and base salts thereof.

Suitable acid addition salts are formed from acids which form non-toxicsalts. Illustrative examples include the acetate, aspartate, benzoate,besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate,citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate,glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate,succinate, tartrate, tosylate and trifluoroacetate salts.

Suitable base salts of the conjugates described herein are formed frombases which form non-toxic salts. Illustrative examples include thearginine, benzathine, calcium, choline, diethylamine, diolamine,glycine, lysine, magnesium, meglumine, olamine, potassium, sodium,tromethamine and zinc salts. Hemi-salts of acids and bases may also beformed, for example, hemi-sulphate and hemi-calcium salts.

In various embodiments of the methods described herein, the conjugatesdescribed herein may be administered alone or in combination with one ormore other conjugates described herein or in combination with one ormore other drugs (or as any combination thereof). In one embodiment, theconjugates described herein may be administered as a formulation inassociation with one or more pharmaceutically acceptable carriers. Thecarriers can be excipients. The choice of carrier will to a large extentdepend on factors such as the particular mode of administration, theeffect of the carrier on solubility and stability, and the nature of thedosage form. Pharmaceutical compositions suitable for the delivery ofconjugates described herein and methods for their preparation will bereadily apparent to those skilled in the art. Such compositions andmethods for their preparation may be found, for example, in Remington:The Science & Practice of Pharmacy, 21th Edition (Lippincott Williams &Wilkins, 2005), incorporated herein by reference.

In one illustrative aspect, a pharmaceutically acceptable carrierincludes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, and combinations thereof, that are physiologically compatible. Insome embodiments, the carrier is suitable for parenteral administration.Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions.

In various embodiments, liquid formulations may include suspensions andsolutions. Such formulations may comprise a carrier, for example, water,ethanol, polyethylene glycol, propylene glycol, methylcellulose or asuitable oil, and one or more emulsifying agents and/or suspendingagents. Liquid formulations may also be prepared by the reconstitutionof a solid, for example, from a sachet.

In one embodiment, an aqueous suspension may contain the conjugatesdescribed herein in admixture with appropriate excipients. Suchexcipients are suspending agents, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents which may be a naturally-occurringphosphatide, for example, lecithin; a condensation product of analkylene oxide with a fatty acid, for example, polyoxyethylene stearate;a condensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethyleneoxycetanol; a condensationproduct of ethylene oxide with a partial ester derived from fatty acidsand a hexitol such as polyoxyethylene sorbitol monooleate; or acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example, polyoxyethylenesorbitan monooleate. The aqueous suspensions may also contain one ormore preservatives, for example, ascorbic acid, ethyl, n-propyl, orp-hydroxybenzoate; or one or more coloring agents.

In one illustrative embodiment, dispersible powders and granulessuitable for preparation of an aqueous suspension by the addition ofwater provide the conjugate in admixture with a dispersing or wettingagent, suspending agent and one or more preservatives. Additionalexcipients, for example, coloring agents, may also be present.

Suitable emulsifying agents may be naturally-occurring gums, forexample, gum acacia or gum tragacanth; naturally-occurring phosphatides,for example, soybean lecithin; and esters including partial estersderived from fatty acids and hexitol anhydrides, for example, sorbitanmono-oleate, and condensation products of the said partial esters withethylene oxide, for example, polyoxyethylene sorbitan monooleate.

In other embodiments, isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride can be included in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, monostearate salts and gelatin.

In one aspect, a conjugate as described herein may be administereddirectly into the blood stream, into muscle, or into an internal organ.Suitable routes for such parenteral administration include intravenous,intraarterial, intraperitoneal, intrathecal, epidural,intracerebroventricular, intraurethral, intrasternal, intracranial,intratumoral, intramuscular and subcutaneous delivery. Suitable meansfor parenteral administration include needle (including microneedle)injectors, needle-free injectors and infusion techniques.

In one illustrative aspect, parenteral formulations are typicallyaqueous solutions which may contain carriers or excipients such assalts, carbohydrates and buffering agents (preferably at a pH of from 3to 9), but, for some applications, they may be more suitably formulatedas a sterile non-aqueous solution or as a dried form to be used inconjunction with a suitable vehicle such as sterile, pyrogen-free water.In other embodiments, any of the liquid formulations described hereinmay be adapted for parenteral administration of the conjugates describedherein. The preparation of parenteral formulations under sterileconditions, for example, by lyophilization under sterile conditions, mayreadily be accomplished using standard pharmaceutical techniques wellknown to those skilled in the art. In one embodiment, the solubility ofa conjugate used in the preparation of a parenteral formulation may beincreased by the use of appropriate formulation techniques, such as theincorporation of solubility-enhancing agents.

In various embodiments, formulations for parenteral administration maybe formulated to be for immediate and/or modified release. In oneillustrative aspect, conjugates of the invention may be administered ina time release formulation, for example in a composition which includesa slow release polymer. The conjugates can be prepared with carriersthat will protect the conjugates against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid and polylactic, polyglycoliccopolymers (PGLA). Methods for the preparation of such formulations aregenerally known to those skilled in the art. In another embodiment, theconjugates described herein or compositions comprising the conjugatesmay be continuously administered, where appropriate.

In one embodiment, sterile injectable solutions can be prepared byincorporating the conjugate in the required amount in an appropriatesolvent with one or a combination of ingredients described above, asrequired, followed by filtered sterilization. Typically, dispersions areprepared by incorporating the conjugate into a sterile vehicle whichcontains a dispersion medium and any additional ingredients from thosedescribed above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the conjugateplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

The composition can be formulated as a solution, microemulsion,liposome, or other ordered structure suitable to high drugconcentration. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. In one embodiment, the proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants.

In one embodiment, compositions described herein comprise a drugdelivery conjugate having a purity of at least 90%. In anotherembodiment, the drug delivery conjugate has a purity of at least 95%. Inanother embodiment, the drug delivery conjugate has a purity of at least96%. In another embodiment, the drug delivery conjugate has a purity ofat least 97%. In another embodiment, the drug delivery conjugate has apurity of at least 98%. In another embodiment, the drug deliveryconjugate has a purity of at least 99%.

The drug delivery conjugates described herein can be prepared byart-recognized synthetic methods. The synthetic methods are chosendepending upon the selection of the optionally addition heteroatoms orthe heteroatoms that are already present on the spacer linkers,releasable linkers, the drug, and/or or the receptor binding ligand (B).In general, the relevant bond forming reactions are described in RichardC. Larock, “Comprehensive Organic Transformations, a guide to functionalgroup preparations,” VCH Publishers, Inc. New York (1989), and inTheodora E. Greene & Peter G. M. Wuts, “Protective Groups ion OrganicSynthesis,” 2d edition, John Wiley & Sons, Inc. New York (1991), thedisclosures of which are incorporated herein by reference. Additionaldetails for preparing functional groups, including amides and esters,ketals and acetals, succinimides, silyloxys, hydrazones, acylhydrazines, semicarbazones, disulfides, carbonates, sulfonates, and thelike contained in the linker, including releasable linkers are describedin U.S. patent application publication No. US 2005/0002942 A1,incorporated herein by reference in its entirety.

General formation of folate-peptides. The folate-containing peptidylfragment Pte-Glu-(AA)_(n)-NH(CHR₂)CO₂H (3) can be prepared by apolymer-supported sequential approach using standard methods, such asthe Fmoc-strategy on an acid-sensitive Fmoc-AA-Wang resin (1), as shownin Scheme 1.

(a) 20% piperidine/DMF; (b) Fmoc-AA-OH, PyBop, DIPEA, DMF; (c)Fmoc-Glu(O-t-Bu)-OH, PyBop, DIPEA, DMF; (d) 1. N¹⁰(TFA)-Pte-OH; PyBop,DIPEA, DMSO; (e) TFAA, (CH₂SH)₂, i-Pr₃SiH; (f) NH₄OH, pH 10.3.

In this illustrative embodiment of the processes described herein, R₁ isFmoc, R₂ is the desired appropriately-protected amino acid side chain,and DIPEA is diisopropylethylamine. Standard coupling procedures, suchas PyBOP and others described herein or known in the art are used, wherethe coupling agent is illustratively applied as the activating reagentto ensure efficient coupling. Fmoc protecting groups are removed aftereach coupling step under standard conditions, such as upon treatmentwith piperidine, tetrabutylammonium fluoride (TBAF), and the like.Appropriately protected amino acid building blocks, such asFmoc-Glu-OtBu, N¹⁰-TFA-Pte-OH, and the like, are used, as described inScheme 1, and represented in step (b) by Fmoc-AA-OH. Thus, AA refers toany amino acid starting material that is appropriatedly protected. It isto be understood that the term amino acid as used herein is intended torefer to any reagent having both an amine and a carboxylic acidfunctional group separated by one or more carbons, and includes thenaturally occurring alpha and beta amino acids, as well as amino acidderivatives and analogs of these amino acids. In particular, amino acidshaving side chains that are protected, such as protected serine,threonine, cysteine, aspartate, and the like may also be used in thefolate-peptide synthesis described herein. Further, gamma, delta, orlonger homologous amino acids may also be included as starting materialsin the folate-peptide synthesis described herein. Further, amino acidanalogs having homologous side chains, or alternate branchingstructures, such as norleucine, isovaline, β-methyl threonine, β-methylcysteine, β,β-dimethyl cysteine, and the like, may also be included asstarting materials in the folate-peptide synthesis described herein.

The coupling sequence (steps (a) & (b)) involving Fmoc-AA-OH isperformed “n” times to prepare solid-support peptide 2, where n is aninteger and may equal 0 to about 100. Following the last coupling step,the remaining Fmoc group is removed (step (a)), and the peptide issequentially coupled to a glutamate derivative (step (c)), deprotected,and coupled to TFA-protected pteroic acid (step (d)). Subsequently, thepeptide is cleaved from the polymeric support upon treatment withtrifluoroacetic acid, ethanedithiol, and triisopropylsilane (step (e)).These reaction conditions result in the simultaneous removal of thet-Bu, t-Boc, and Trt protecting groups that may form part of theappropriately-protected amino acid side chain. The TFA protecting groupis removed upon treatment with base (step (f)) to provide thefolate-containing peptidyl fragment 3.

In each of the foregoing synthetic processes, the intermediates may becoupled with any additional hydrophilic spacer linkers, other spacerlinkers, releasable linkers, or the therapeutic agent A. In variationsof each of the foregoing processes, additional hydrophilic spacerlinkers, other spacer linkers, or releasable linkers may be insertedbetween the receptor binding ligand B and the indicated hydrophilicspacer linkers. In addition, it is to be understood that theleft-to-right arrangement of the bivalent hydrophilic spacer linkers isnot limiting, and accordingly, the therapeutic agent A, the receptorbinding ligand B, additional hydrophilic spacer linkers, other spacerlinkers, and/or releasable linkers may be attached to either end of thehydrophilic spacer linkers described herein.

METHOD EXAMPLES Example Adjuvant-Induced Arthritis (AIA) Model

Female Lewis rats were fed a folate-deficient diet (Harlan Teklad,Indianapolis, Ind.) for 9-10 days prior to arthritis induction. Theadjuvant-induced arthritis (AIA) was induced by intradermal inoculation(at the base of tail) of 0.5 mg of heat-killed Mycobacteria butyricum(BD Diagnostic Systems, Sparks, Md.) in 100 μL light mineral oil(Sigma). Ten days after arthritis induction, paw edema in rats wasassessed using a modified arthritis scoring system: 0=no arthritis;1=swelling in one type of joint; 2=swelling in two types of joint;3=swelling in three types of joint; 4=swelling of the entire paw. Atotal score for each rat is calculated by summarizing the scores foreach of the four paws, giving a maximum score of 16 for each rat. On Day10 post arthritis induction, rats with a total arthritis score of ≥2were removed from the study and the remaining rats were distributedevenly across the control and treatment groups (n=5 for all groupsexcept that n=2-3 for healthy controls). All treatments started on Day10 unless mentioned otherwise.

Example EC0746 Demonstrated FR-Mediated Inhibition of DHFR, Viability,and LPS-Stimulated TNF-α Production in RAW264.7 Cells

RAW264.7 cells were treated with vehicle (medium), EC0746 (100 nM)without or with 100-fold excess free folate, aminopterin (AMT, 100 nM),methotrexate (MTX, 100 nM), or excess free folate alone (10 μM). After 1h incubation, the drug-containing media were replaced with fresh mediumand the cells were allowed to incubate further for 24 h. At the end ofincubation, the cells were lysed and the DHFR activity in cell lysateswas measured using a commercial DHFR assay kit (Sigma-Aldrich, SaintLouis, Mo.). See FIG. 1.

For XTT cell viability and TNF-α inhibition assays. RAW264.7 cells in96-well plates were treated with vehicle (culture medium) or 10-foldserial dilutions of EC0746 without or with 100-fold excess free folate.After 2 h incubation, the drug-containing media were replaced and thecells were allowed to incubate further for 70 h. Four hours prior to theend of incubation, LPS was added to the treated cells at a finalconcentration of 100 ng/mL. 100 μL of the culture supernatants werecollected for TNF-α analysis using a commercial ELISA kit. See FIG. 2B.The cell viability was assessed by adding XTT(2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide)to the remaining medium for an additional 4 h following themanufacturer's instructions (Roche Applied Science, Indianapolis, Ind.)See FIG. 2A. Both results were expressed as % absorbance (minusbackground) relative to untreated control in triplicates. The resultsdemonstrated that EC0746 inhibited the viability of RAW264.7 cells andthe ability of these cells to produce TNF-α in response to LPS.

Example EC0746 Inhibited Lps-Stimulated Cytokine Production fromThioglycollate-Elicited Macrophages in a Fr-Dependent Manner

To obtain thioglycollate-elicited macrophages, female Lewis rats weredosed once intraperitoneally with an aged thioglycollate medium (20ml/kg) and euthanized 3 days later. The peritoneal cavity of the animalswas lavaged with 60-70 ml of ice-cold PBS buffer to collect peritonealextrudate. Thioglycollate-elicited macrophages in the peritoneal fluidswere obtained after a red cell lysing step and a 2-hour adherence incell culture medium containing 1% heat-inactivated fetal bovine serum.

Rat thioglycollate-elicited macrophages were treated with medium only,methotrexate (100 nM), aminopterin (100 nM), EC0746 (100 nM) without orwith 100-fold excess free folate (10 μM), or excess free folate alone(10 μM). The drug-containing media were removed after 2 h incubation andthe cells were allowed to incubate further for an additional 70 h infresh medium. Twenty-four hours prior to the end of incubation, LPS (5μg/mL) and IFN-γ (100 ng/mL) were added to the above cells to stimulatecytokine production. Cytokines (TNF-α, IL-1α, IL-6, IL-10, MIP-1α, etc.)released into the cell culture medium were measured using a rat cytokinearray assay kit (R&D Systems, Minneapolis, Minn.). See FIG. 3.

Example EC0746 Treatment Reduced Local (Paw) and Systemic (Spleen)Inflammation in Rats with Adjuvant Arthritis

Rats with adjuvant arthritis were treated subcutaneously with EC0746(500 nmol/kg) on days 10, 13, 17, and 20 post arthritis induction. Theanimals in the healthy and arthritis control groups were left untreated.The arthritis scores and animal body weights were recorded three times aweek (see FIGS. 4A, B). At the completion of study (days 24-25), therats were euthanized by CO₂ asphyxiation and processed for paw andspleen weights. The results showed that EC0746-treatment rats hadsignificantly less arthritis score, paw weight (i.e. paw swelling), andspleen weights (see FIGS. 5A, B). The overall reduction of local andsystemic inflammation in the EC0746-treated rats rendered these animalsbetter body weight compared to animals in the untreated arthritiscontrol group.

Example EC0746 Treatment Prevented Bone Damage in Rats with AdjuvantArthritis

Rats with adjuvant arthritis were treated subcutaneously with EC0746(500 nmol/kg) on days 10, 13, 17, and 20 post arthritis induction. Theanimals in the healthy and arthritis control groups were left untreated.At the completion of study, rat hind paws were photographed and theanimals were euthanized by CO₂ asphyxiation. X-ray radiographic imagesof hind paws were taken immediately using a Kodak Imaging Station InVivo FX (Carestream Molecular Imaging, New Haven, Conn.). Therepresentative X-ray images of hind paws were shown for a healthy ratand EC0746-treated or untreated arthritic rats. Compared to severe boneerosion seen with the untreated arthritis control animal, EC0746treatment starting at the on-set of arthritis development (day 10)effectively halted paw swelling/inflammation and prevented bone erosion.See FIGS. 6A, B.

Example EC0746 was as Effective as Mtx at Equal Molar Subcutaneous Doses

Rats with adjuvant arthritis were treated subcutaneously with EC0746(300 nmol/kg) or methotrexate (MTX, 300 nmol/kg) on days 10, 13, 17, and20 post arthritis induction. The animals in the healthy and arthritiscontrol groups were left untreated. The arthritis score and animal bodyweight were recorded three times a week (see FIGS. 7A, B). At thecompletion of study, the rats were euthanized by CO₂ asphyxiation andprocessed for paw and spleen weights. The results showed that biweeklysubcutaneously dosed EC0746 and methotrexate were similarly active inreducing local (paw) and systemic (spleen) inflammation in thesearthritic animals. See FIGS. 8A, B.

Example Anti-Arthritis Activity of EC0746 (but not MTX) could bePartially Blocked by a Co-Injected Folate Competitor

Rats with adjuvant arthritis were treated subcutaneously with EC0746(500 nmol/kg) or MTX (500 nmol/kg) in the absence or presence of300-fold excess of Re-EC0589 (150 μmol/kg, Rhenium complex of EC0589) orRe-EC20 (150 μmol/kg, Rhenium complex of EC20), respectively, on days10, 13, 17, and 20 post arthritis induction. The arthritis score andanimal body weight were recorded three times a week. Both EC0589 andRe-EC20 served as a FR-binding competitor. The animals in the arthritiscontrol group were left untreated. The results showed that theanti-arthritis activity of EC0746 but not MTX could be partially blockedby excess amount of a FR-binding competitor.

Example Collagen-Induced Arthritis (CIA) Model

The collagen-induced arthritis (CIA) was induced in female Lewis rats onfolate-deficient diet (Harlan Teklad, Indianapolis, Ind.). On Day 0,rats were immunized with 500 μg of bovine collagen Type II (Chondrex,Redmond, Wash.) formulated with Freund's complete adjuvant. A boosterimmunization was given on Day 7 with 250 μg of the bovine collagenformulated with Freund's incomplete adjuvant. Arthritis disease wasassessed by a qualitative clinical score system described by themanufacturer (Chondrex, Redmond, Wash.): 0=normal, 1=Mild, but definiteredness and swelling of the ankle or wrist, or apparent redness andswelling limited to individual digits, regardless of the number ofaffected digits, 2=Moderate redness and swelling of ankle of wrist,3=Severe redness and swelling of the entire paw including digits, and4=Maximally inflamed limb with involvement of multiple joints. On Day 10post first immunization, rats were distributed evenly (according to thearthritis score) across the control and treatment groups. The CIA ratswere given ten consecutive subcutaneous doses of EC0746 and methotrexateon days 10-19. For both drugs, an induction dose (500 nmol/kg) was givenon days 10 and 15 and a maintenance dose (100 nmol/kg) was given on days11-14 and 16-19. The animals in the arthritis control group were leftuntreated. The arthritis score and animal body weight were recorded fivetimes a week. The result showed that EC0746 was also effective in ratswith collagen-induced arthritis. See FIGS. 13A, B.

Example EC0746 Plasma Pharmacokinetics after a Single S.C. Dose

Female Lewis rats with rounded tip jugular vein catheters (Harlan) werefed regular rodent diet and used in this study. The animals were given asingle subcutaneous dose of EC0746 at 500 nmol/kg. Whole blood samples(300 μl) were collected from the animals at the following time points: 1min, 10 min, 30 min, 1h, 2h, 3h, 4h, and 8h after injection. The bloodsamples were placed into anti-coagulant tubes containing 1.7 mg/mL ofK3-EDTA and 0.35 mg/mL of N-Maleoyl-beta-alanine (0.35 mg/mL). Plasmasamples were obtained by centrifugation for 3 min at ˜2,000 g and storedat −80° C. The amount of EC0746 and its released base drugs (EC0470 &aminopterin) were determined by HPLC using the EC0746 injection solutionas the standard (see FIG. 14). The result showed that approximately 18%of free drug exposure/release (EC0470 & aminopterin) was detected in theplasma after a single subcutaneous dose of EC0746. However, the Tmax ofEC0746 was observed at ˜30 min while EC0470 and aminopterin showed adelayed Tmax at ˜1 h. See FIG. 14. A similar method was used todetermine the plasma pharmacokinetics of aminopterin (see FIG. 15).

Example Maximum Tolerated Dose (MTD) of Aminopterin and EC0746

Healthy rats were administered a subcutaneous injection of the indicateddose of aminopterin or EC0746 biweekly for 2 weeks; control animals, notreatment, 100 nmole/kg aminopterin, 50 nmole/kg aminopterin, 500nmole/kg EC0746, or 2000 nmole/kg EC0746. The animals were weigheddaily. See FIGS. 16A, B. A dose of 0.1 μmol/kg of aminopterin in folatedeficient rats is above the MTD; therefore, the projected MTD of EC0746would be <0.5 μmol/kg based solely on ˜20% free drug release shown inthe previous Example. However, the MTD of EC0746 is actually 2.0μmol/kg, which is equivalent to 0.4 μmol/kg, or ˜8× higher than the MTDfor free aminopterin. On a molar basis for total aminopterin, the MTD is40× higher. The therapeutic index (MTD for healthy animals/ED₅₀ fortreatment of adjuvant-induced arthristis in female Lewis rats) for EC746is about 9.5 (2000/210).

Example Mechanism of Action: EC0932 as a Folate-Targeted Antifolate

RAW264.7 cells in 96-well plates were treated with vehicle (culturemedium) or 10-fold serial dilutions of EC0932 without or with 100-foldexcess free folate. The drug-containing medium was replaced after 2 htreatment and the cells were allowed to incubate further in standardmedium for 70 h. Four hours prior to the end of incubation, LPS wasadded to the treated cells at a final concentration of 100 ng/mL. 100 μLof the culture supernatants were collected for TNF-α analysis using acommercial ELISA kit (see FIG. 17B). The cell viability was assessed byadding XTT(2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide)to the remaining medium for an additional 4 h following themanufacturer's instructions (Roche Applied Science, Indianapolis, Ind.).See FIG. 17A. Both results were expressed as % absorbance (minusbackground) relative to untreated control in triplicates. The resultsdemonstrated that EC0932 inhibited the viability of RAW264.7 cells andtheir ability to produce TNF-α in response to LPS.

Example Mechanism of Action: EC0932 as a Folate-Targeted mTOR Inhibitor

RAW264.7 cells were treated with medium only (UTC), 100 nM everolimus(mTOR inhibitor), 100 nM aminopterin (antifolate), 100 μM EC0823 (C, aFR-binding competitor), or 100 nM EC0932 without or with 100 μM ofEC0823. The drug-containing media were removed after 2 h and the cellswere allowed to incubate for 4 h in fresh medium. Afterwards, the celllysates were collected and subjected to Western blot analysis forphosphorylation of S6 ribosomal protein (p-RPS6), a downstream target inthe mTOR signaling pathway. The data showed that EC0932 treatmentresulted in down-regulation of p-RPS6 and its inhibitory effect could bepartially blocked by excess EC0823, a FR-binding competitor. See FIG.18.

Example EC0932 Demonstrates FR-Specific Activity Against AdjuvantArthritis

Rats with adjuvant arthritis were treated subcutaneously with EC0932(250 nmol/kg) in the absence or presence of 500-fold excess of EC0923(125 μmol/kg) on days 10, 13, 17, and 20 post arthritis induction. Theanimals in the healthy and arthritis control groups were left untreated.The arthritis scores and animal body weights were recorded three times aweek (see FIGS. 19A, B). At the completion of study (day 24), the ratswere euthanized by CO₂ asphyxiation and processed for paw and spleenweights (see FIGS. 20A, B). The results showed that the anti-arthritisactivity of EC0932 was blocked by EC0923, a folate competitor withsimilar affinity as free folic acid.

Example EC0828 Demonstrates FR-Specific Activity Against AdjuvantArthritis

Using similar methods to those herein described, the effect of EC0828 onarthritis was measured. See FIGS. 21-24.

Example Relative Folate Receptor Affinity

Using displacement of ³H-folic acid from folate-receptor-α positive KBcells the relative folate affinity (FA) for several compounds wasmeasured. Folic acid (1.0), aminopterin (0.008), methotrexate (0.018),EC0746 (0.5), and EC0932 (0.26). See FIGS. 25A-D.

Using displacement of ³H-folic acid from folate-receptor-beta positiveCHO-FR-β cells, the relative folate affinity (FA) for several compoundswas measured. Folic acid (1.0), aminopterin (0.004), methotrexate(0.005), and EC0746 (0.27). See FIG. 25F.

Example EC0746 is a High-Affinity FR-Specific DHFR Inhibitor

The FR-binding affinity of EC0746 was directly compared to that ofaminopterin (AMT) and methotrexate (MTX) in a competitive binding assayusing KB cells as the source of FR. ³H-folic acid was used as thecompetitive ligand and the relative affinity of folate itself was setto 1. As shown in FIGS. 25A-D, EC0746 displayed a much higher affinityto KB cells (FR-α positive) than AMT and MTX with relative affinityvalues of 0.50, 0.004, and 0.018 respectively. To demonstrateFR-specific activities in-vitro, we first tested the ability of EC0746to inhibit DHFR, an intracellular target involved in cellular division.FR-positive RAW264.7 cells were given a 2-h pulse of 100 nM EC0746without or with 100-fold excess folic acid (folate competition) followedby a 22-h “chase” in a drug-free medium. As shown in FIG. 25E, EC0746inhibited DHFR activity in RAW264.7 cells to a similar degree as AMT andMTX, but excess folic acid completely abolished its inhibitory effect.Since excess folic acid alone (10 μM) did not have any impact, our datasuggested that EC0746 may be similar to AMT and MTX with regard to DHFRinhibition but its activity is FR-specific.

Example EC0746 Demonstrates an Anti-Proliferative Effect on RAW264.7Cells

To study the anti-proliferative effect of EC0746, RAW264.7 cells (˜40%confluency) were given a 2 h treatment of serial dilutions of EC0746without or with excess folic acid and followed by a 70-h “chase” indrug-free medium. At 4 h before the end of incubation, LPS (100 ng/ml)was added to the culture medium to stimulate cytokine production. Asdetermined by the XTT assay (FIG. 26A), EC0746 showed a dose-dependentinhibition of cell proliferation (ED₅₀≈0.3 nM); however, the maximumeffect was ˜50% when compared to the untreated cells. The EC0746-treatedRAW264.7 cells produced less TNF-α upon LPS exposure (ED₅₀≈1.6 nM) (FIG.26B). The anti-proliferative and anti-TNF activities of EC0746 were 100%competable with excess folic acid. Interestingly, RAW264.7 cells thathad “survived” the EC0746 treatment at ≥1 nM concentrations showed nosign of additional growth when redispersed in fresh medium for 72 h.Because DHFR is an S-phase enzyme that increases during the S-phase ofmitosis, RAW264.7 cells pre-treated with EC0746 were stained withpropidium iodide and analyzed for the status of cell cycle and theexpression of proliferating cell nuclear antigen (PCNA). For flowcytometric analysis (FACS) of the cell cycle, the cells were recovered,fixed in 70% ethanol/PBS solution, and resuspended in FACS buffer (PBSsupplemented with 1% BSA). After a brief treatment with RNase A (RocheMolecular Biochemicals), the cells were stained with 50 μg/ml ofpropidium iodide (Invitrogen, Carlsbad, Calif.) before FACS analysis.Western blot analysis was carried out on whole cell lysates using amonoclonal antibody specific for PCNA (PC10, Cell Signaling, Danvers,Mass.). After incubation with a peroxidase-conjugated secondaryantibody, the signals were visualized using SuperSignal West PicoChemiluminescent Substrate system (ThermoScientific, Waltham, Mass.)following the manufacturer's instructions. The images were acquiredusing a G:BOX Chemi HR 16 gel imaging system (Syngene, Frederick, Md.).As shown in FIGS. 26C-F, EC0746 treated RAW264.7 cells showed anincrease in number of cells in the S-phase, but the effect was againcompetable by excess folic acid. Western blot analysis indicated nochange in PCNA expression in EC0746-treated and untreated cells (FIG.26G). Taken together, these data demonstrated that EC0746 completelyhalted the proliferation of RAW264.7 cells in a FR-dependent manner, butdid not appear to kill them; instead, the cells were arrested at theS-phase of the cell cycle.

Example EC0746 Modulates Cytokine Responses in RatThioglycollate-Elicited Macrophages

Because rat TG-macs are responsive to inflammation stimuli in-vitro, weexamined the ability of EC0746 to block cytokine response after exposureto LPS and IFN-γ, two signals required for full activation ofmacrophages. Thus, rat TG-macs were treated with 100 nM of EC0746following our standard condition of 2 h pulse plus a 70-h chase andwithout or with folate competition. At 24 h prior to the end ofincubation, LPS (5 μg/mL) and IFN-γ (100 ng/mL) were added to theculture medium to stimulate the release of cytokines, chemokines, andother inflammatory mediators. As detected with a rat cytokine antibodyarray (FIG. 27A, B, C), EC0746 inhibited a range ofcytokines/chemokines, 11 of which showed a significant FR-specificinhibition (P<0.05, EC0746 versus EC0746 plus folic acid), includingIL-1β, IL-1γa, MIP-1α, TNF-α, VEGF, CINCs, sICAM, LIX, L-selectin, andMIG. These data also indicated that (i) the levels of FRs on rat TG-macswere sufficient for EC0746 to take a remedial effect on cytokineresponses associated with macrophage activation and (ii) the observedanti-inflammatory action of EC0746 can be independent of macrophageproliferation.

Example EC0746 Ameliorates Local and Systemic Inflammation in Rats withAdjuvant Arthritis

The rat adjuvant induced arthritis (AIA) model resembles thecharacteristics of RA in humans and has been widely used to study theimpact of novel anti-inflammatory agents. In this study, AIA rats withearly-onset or established disease (mean arthritis scores of 0 and 2,respectively) were given a biweekly regimen of EC0746 (s.c., 500nmol/kg/dose) for 2-week starting on day 10-13 after arthritisinduction. Multiple study endpoints were taken to assess the efficacyincluding (i) arthritis score and body weight obtained during the courseof therapy and (ii) paw and spleen weights collected 4 days after thelast dose. As measured by the semiquantitative visual scoring system,EC0746 was found to be very effective in rats with low arthritis at thefirst day of treatment (FIG. 28A). In rats with more established AIA,the same treatment improved, although to a lesser degree, the overallseverity of the joint disease. While the untreated arthritic controlslost approximately 14-16% of their original weight, the animals treatedwith EC0746 from the early-onset maintained a good body weight (FIG.28B). On the other hand, EC0746 did not stop the animals with moreestablished arthritis at the time of treatment from losing weight, butto a lesser extent in relation to the untreated arthritic controls. Whenthe percent increase in paw/spleen weights was evaluated (FIG. 28C),EC0746 treatment resulted in approximately 10-(paw) and 3-fold (spleen)improvements in rats with low arthritis, and the correspondingimprovements in rats with more established arthritis were at ˜2.5- and2-fold, respectively (see P values in the figure legend).

To further investigate dose-response relationship andschedule-dependency, EC0746 was administered to rats with developing AIA(day 10) at 25-500 nmol/kg/dose (biweekly) or 1000 nmol/kg/dose (onceweekly). As summarized in Table 1, below, biweekly EC0746 dosed for 2weeks displayed a linear dose response in inhibiting paw edema from 25to 250 nmol/kg/dose with an R-squared value of 0.997, and no statisticaldifferences between 250 and 500 nmol/kg/dose were seen. When dosed onceweekly for 2 weeks, EC0746 at 1000 nmol/kg/dose remained to be highlyeffective, but this schedule did not control the fast progressing AIA tothe same degree as the biweekly regimen at 250-500 nmol/kg/dose.Overall, EC0746 was found very effective against AIA and capable ofcontrolling local (joints) and systemic (spleen) inflammation andhalting the progression of arthritis.

TABLE 1 % Dose Inhibition (nmol/ in paw % Weight Treatment kg) Frequencyedema¹ Splenomagly² loss³ Control — — 0 117 ± 22 −16 ± 1 EC0746  25* biw 0 ± 25* 73 ± 8 −17 ± 1 (sc)  100* biw 35 ± 11*  52 ± 13 −14 ± 1  250*biw 91 ± 4* 25 ± 6 −0.5 ± 3  500 biw 91 ± 9 37 ± 7  0.4 ± 4 1000  qw 72± 12 39 ± 5  −7 ± 3 MTX 250 biw 70 ± 5  42 ± 17 −14 ± 2 (oral) 1650  qw47 ± 10^(†) — −10 ± 2 MTX (sc) 250 biw 78 ± 10 24 ± 3  −2 ± 4 500 biw 88± 7 20 ± 4  −2 ± 7 Enbrel 10 mg/ q3d 46 ± 9 42 ± 7 −15 ± 2 (sc) kg ¹%Inhibition in paw edema is calculated based on paw weight on Day 24: 100× (Arthritic control - Treated)/(Arthritic control - Healthy).²Splenomagly is defined as % increase in spleen weight relative to thespleen weights of healthy rats. ³On Day 24 relative to body weight onthe first day of treatment. *Linear regression analysis showed anR-squared value of 0.997. ^(†)Calculated based on arthritis scores onDay 24 (paw weights were not obtained).

Example EC0746 Anti-Arthritic Activity was FR-Mediated and Differentfrom MTX

RA patients receiving MTX are frequently given folate supplementation toreduce its side effects. We found simply mixing AMT with free folic acid(1:1) to match the dose of EC0746 (500 nmol/kg/dose, biweekly) was 100%lethal to AIA rats. Thus, the anti-arthritic effect of EC0746 in AIArats was not due to the apparent “folate supplementation of AMT”. Giventhe difference in FR-binding affinity, we suspected that EC0746 and itsactive comparator MTX would have a different mechanism of action in theAIA model where FR-positive macrophages are abundant. To verify thistheory, we undertook in-vivo folate competition studies using EC0923, awater-soluble folate-containing competitor (relative affinity value of0.84 on KB cells), as a FR-blocking agent in the AIA rats. All testcompounds (EC0746, 250 nmol/kg/dose; MTX, 250 nmol/kg/dose; EC0923, 125μmol/kg/dose) were administered biweekly for 2 weeks. EC0923 was pre-and co-administered at a total of 500-fold excess of EC0746 and MTX.

As shown in FIG. 29A, B, C, D, E, F, EC0746 alone was very effective,but its activity could be nearly completely blocked by the presence ofexcess EC0923. This included all parameters assessed: arthritis score(FIG. 29A), change in body weight (FIG. 29B), and percent increases inpaw and spleen weights (FIG. 29C, D) (see P values in the figurelegend). EC0923 treatment alone did not have any impact on thedevelopment or severity of the disease compared to the untreatedarthritic controls (FIG. 29A, B, C, D, E, F). Radiographic analysis(hind paws) confirmed severe periarticular soft tissue swelling, jointspace narrowing, bone erosion, periostal new bone formation, andosteolysis in arthritic control animals and the animals treated withEC0923 (FIGS. 29E, F). There were minimum radiographic changes seen inEC0746-treated animals, but the animals treated with EC0746 plus EC0923showed a significant joint damage (P<0.05). Histologically, animalstreated with EC0923 alone were also similar to untreated arthriticcontrols in all parameters assessed (i.e. ankle inflammation, boneresorption, pannus formation, and cartilage damage) (FIG. 30A). As shownin FIG. 30B, the mean sum of histology scores (out of a maximum score of20) was ˜15.3 and 14.2 for the arthritic control and EC0923-treatedanimals, respectively. The corresponding dorsal to ventral paw thicknesswas ˜9.8 mm and ˜9.4 mm, compared ˜5.8 mm for the healthy animals (FIG.30C). In contrast, 3 of 5 animals treated with EC0746 had no lesions(FIG. 30D), resulting in 88-100% decreases in individually scoredparameters (FIG. 30A) and an overall 94% decrease in summed scores whencompared to untreated arthritic controls (FIG. 30B). The dorsal toventral thickness of EC0746-treated paws was also significantlydecreased by 94% (FIG. 30C). The animals treated with EC0746 plus the500-fold excess EC0923 did have significantly decreased inflammationscores (24%), but all other scored parameters were non-significantlydecreased (9-21%, FIGS. 30A, B). The dorsal to ventral paw thickness inEC0746/EC0923-treated paws was also significantly decreased by 22% fromthat of the arthritic control animals (FIG. 30C), suggesting a small nonFR-targeted effect. On an equimolar basis, s.c. MTX treatment (250nmol/kg/dose, 4 dose×2 weeks) also significantly improved thedevelopment and severity of arthritis (Table 1, above, FIG. 31A, B, C).However, the anti-arthritic activity of MTX was not significantlyblocked by excess EC0923 in arthritis score (FIG. 31A) and percentincreases in paw and spleen weights (FIG. 31B). The animals treated withMTX plus the folate competitor did actually lose more body weightcompared to the animals treated with MTX (FIG. 31C). Taken together, ourdata suggested that EC0746 and MTX are different from each other interms of treating active inflammation via FR-targeted and non-targetedmechanisms of action, respectively.

Example EC0746 is More Efficacious than Oral Methotrexate andSubcutaneous Etanercept

Since methotrexate (MTX) and etanercept are part of the current standardof care for RA, we compared EC0746 against both drugs in rats withadjuvant arthritis using clinically relevant dosing routes. The animalswere treated with subcutaneous EC0746 (250 nmo/kg) and oral MTX (250nmol/kg) on days 10, 13, 17, and 20 post arthritis induction. Etanerceptwas given subcutaneously at 10 mg/kg once every 3 days starting on day10. At the completion of any study (day 24), rats were euthanized by CO₂asphyxiation and processed for paw weight (cut at the hairline) andspleen weight. The differences in total paw weight between arthriticrats after treatment and that of healthy rats from the same experimentwere used to verify the extent of paw edema. The removed hind paws wereimmersion-fixed in 10% buffered formalin and subjected to radiographicand/or histopathological analyses. In some cases, X-ray radiographicimages of the arthritic hind paws were taken using a Kodak ImagingStation In Vivo FX system (Carestream Molecular Imaging, New Haven,Conn.).

As shown in FIG. 29A, B, C, D, E, F and Table 1, above, subcutaneouslyadministered EC0746 (250 nmol/kg/dose, 4 doses×2 weeks) was found moreefficacious than oral MTX on an equimolar basis in most clinical andradiographic parameters assessed: arthritis score (FIG. 29A), change inbody weight (FIG. 29B), percent inhibition in paw edema (Table 1, above,calculated from FIG. 29C) and radiographic score (FIGS. 29E, F).Although EC0746 and MTX-treated arthritic animals had significantlydecreased spleen weight compared to arthritic controls, they were notstatistically different from each other (FIG. 29D, Table 1, above). Inthe same study, EC0746 was found more effective than etanercept in allparameters assessed except for the spleen. Histological grading ofarthritis compared to the untreated controls showed oral MTX-treatedanimals had significant reductions (66-84%) in all scored parameters(i.e. ankle inflammation, bone resorption, pannus formation, andcartilage damage (FIG. 30A). There was a 73% significant decrease in thesummed histological score (FIG. 30B). While etanercept was not aseffective as oral MTX, animals treated with etanercept also hadsignificant reductions in inflammation (42%) and bone resorption (55%),which contributed to a significant 43% decrease in the summed score(FIGS. 30A, B). The dorsal to ventral paw thicknesses in both MTX andetanercept-treated rats were significantly decreased by 63% and 40%,respectively (FIG. 30C). Overall, EC0746 consistently outperformed bothoral MTX and s.c. etanercept in all histological parameters assessed,with further decreases in the summed scores and dorsal to ventral pawthicknesses.

Example EC0746 is Less Toxic than Aminopterin and Methotrexate inFolate-Deficient Rats

Since the toxicity of antifolates can be easily masked by rodent dietsenriched with folate, healthy rats on a folate-deficient diet (Harlan)were used to determine the maximum-tolerated-dose levels (MTD) ofEC0746, AMT and MTX. The animals were given biweekly injections ofEC0746, AMT, and MTX for two weeks. The MTD dose was defined as the dosethat had caused at least 13-14% weight loss combined with clinical signsof stress and at least one animal in the >MTD dose group needed to beeuthanized. Standard hematologic and blood chemistry parameters wereexamined as needed along with histopathology. The MTDs of EC0746, MTXand AMT were determined to be 2000, 1000, and 50 nmol/kg, respectively.At above the MTD dose level, the main toxicities of EC0746 were similarto those of AMT and MTX, including manifested gastrointestinal distress(diarrhea), swollen muzzle, immunosuppression (bone marrow, thymus), lowwhite-blood-cell count, low platelet count, and infections. Whileimmunosuppression is the dose-limiting toxicity of all three of thesecompounds, EC0746 at its MTD dose in rats showed lessgastrointestinal-associated toxicities than AMT and MTX. Overall, EC0746was approximately 40-fold less toxic than AMT and 2-fold less toxic thanMTX on an equimolar basis in these folate-deficient animals; however,its toxicity profile at above MTD levels was not dissimilar from that ofAMT and MTX.

Example EC0746 Pharmacokinetics in Rats

EC0746 is bioavailable after subcutaneous administration in rats and hasa serum protein binding of ˜46%. Both AMT and AMT hydrazide areanticipated metabolites because EC0746 contains ahydrazide/disulfide-based releasable linker. Notably, AMT hydrazide andAMT are equally potent on RAW264.7 cells by inhibiting cellproliferation (FIG. 32A) and LPS-stimulated TNF-α production (FIG. 32B).Thus, the plasma concentrations of EC0746 and two primary metabolites,AMT and AMT hydrazide, were determined by LC/MS/MS after a singlesubcutaneous EC0746 administration. As shown in FIG. 33A, subcutaneouslyadministered EC0746 (500 nmol/kg) reached the blood stream withinminutes, peaked around 10-30 min, and maintained a plateau until 60minutes. EC0746 was cleared rapidly from the blood with an eliminationhalf-life of ˜35 min. Interestingly, the peak appearances of AMT and AMThydrazide in the plasma were nearly superimposable in the EC0746-dosedrats with a 30-min delay from the EC0746 Cmax. For comparison, thepharmacokinetics of subcutaneously dosed AMT (500 nmol/kg) was alsoexamined (FIG. 33B). The AMT Cmax was more similar to that of EC0746than to those of EC0746-derived AMT/AMT hydrazide seen in FIG. 33A.However, the elimination half-life of subcutaneously administered freeAMT was ˜140 min, more similar to that of AMT (˜117 min) and AMThydrazide (˜187 min) released from EC0746. Based onarea-under-the-curve, ˜18% of active drug exposure/release (AMT plus AMThydrazide) was detected in the plasma over 8 h collection period in theEC0746 dosed animals (FIG. 33C).

Example Animal Experimental Autoimmune Uveitis Model

Experimental autoimmune uveitis (EAU) was induced in female Lewis ratsmaintained on a folate-deficient diet (Harlan Teklad, Indianapolis,Ind.). On Day 0, the animals were immunized subcutaneously with 25 μg ofbovine S-Ag PDSAg peptide formulated with Freund's incomplete adjuvantcontaining 0.5 mg of M. Tuberculosis H37Ra. Purified pertussis toxin(PT) was given at a dosage of 1 μg per animal on the same day viaintraperitoneal injection. The severity of uveitis in each eye wasassessed by a qualitative visual score system: 0=No disease, eye istranslucent and reflects light (red reflex); 0.5 (trace)=Dilated bloodvessels in the iris, 1=Engorged blood vessels in iris, abnormal pupilcontraction; 2=Hazy anterior chamber, decreased red reflex; 3=Moderatelyopaque anterior chamber, but pupil still visible, dull red reflex; and4=Opaque anterior chamber and obscured pupil, red reflex absent,proptosis. This assessment yields a maximum uveitis score of 8 peranimal. FIG. 34 shows images the eyes of an animal (upper right) withsevere uveitis on its right eye (bottom) and a healthy eye (upperright).

Example EC0746 Treatment Effectively Reduced EAU Inflammation

Animals treated according to the preceding method to induce EAU wererandomized and distributed into two groups: (1) the untreatedexperimental autoimmune uveitis control group and (2) the EC0746 treatedexperimental autoimmune uveitis group. The animals in the experimentalautoimmune uveitis control group were untreated. The animals in theEC0746 treatment group were given subcutaneous doses of EC0746 at adosage of 500 nmol/kg every other day starting on day 7 after EAUinduction. The uveitis score and animal body weight were recorded foreach animal on days 7-9 and 11-15, see FIG. 35 (the uveitis score,calculated as described in the preceding example, is shown). On day 19,the animals were euthanized and the aqueous humor samples were collectedfrom the anterior chamber for total protein analysis (see FIG. 37).Increased protein levels in aqueous humor are symptomatic of ocularinflammation.

Example Adjuvant-Induced Arthritis (AIA) Model

Female Lewis rats were fed a folate-deficient diet (Harlan Teklad,Indianapolis, Ind.) for 9-10 days prior to arthritis induction. Theadjuvant-induced arthritis (AIA) was induced by intradermal inoculation(at the base of tail) of 0.5 mg of heat-killed Mycobacteria butyricum(BD Diagnostic Systems, Sparks, Md.) in 100 μL light mineral oil(Sigma). Ten days after arthritis induction, paw edema in rats wasassessed using a modified arthritis scoring system: 0=no arthritis;1=swelling in one type of joint; 2=swelling in two types of joint;3=swelling in three types of joint; 4=swelling of the entire paw. Atotal score for each rat is calculated by summarizing the scores foreach of the four paws, giving a maximum score of 16 for each rat. On Day10 post arthritis induction, rats with a total arthritis score of ≥2were removed from the study and the remaining rats were distributedevenly across the control and treatment groups (n=5 for all groupsexcept that n=2-3 for healthy controls). All treatments started on Day10 unless mentioned otherwise.

Example EC0565 Mediated Fr-Specific Inhibition of mTOR Signaling inMacrophages

To examine the targeting effect of EC0565 on FR-positive macrophages,RAW264.7, thioglycolate-elicited macrophages (TG-macs), and arthriticmacrophages from AIA rats (AIA-macs) were treated with medium only(UTC), everolimus (10 and 100 nM), EC0565 (1, 10, 30, and 100 nM), orEC0565 (1, 10, 30, and 100 nM) plus 100 μM excess of a folate competitor(EC17 or free folate). The drug-containing media were removed after 1 hand the cells were allowed to incubate from 6 h in fresh medium.Afterwards, the cell lysates were collected and subjected to Westernblot analysis for phosphorylation of S6 ribosomal protein (p-RPS6), adownstream target in the mTOR signaling pathway.

EC0565 treatment resulted in down-regulation of p-RPS6 at nanomolarconcentrations in all macrophages tested (see FIGS. 38A-C). EC0565appeared to be less potent than everolimus (see FIG. 38B), but itsinhibitory effect was dose dependent (see FIG. 38C) and mediated by theFR (see FIGS. 38A-C). The presence of excess EC17 (see FIG. 38A, afolate-containing ligand) or free folic acid (see FIGS. 38B, C) reversedthe effect EC0565 on these cells. More importantly, despite the lower FRexpression in TG-macs and AIA-macs than in RAW264.7 cells, these resultssuggested that the amount of FRs on these ex-vivo isolated macrophageswere sufficient to deliver a FR-specific target inhibition of themTOR-signaling pathway.

Example Subcutaneously Dosed EC0565 and Everolimus were Similarly Active

AIA rats were treated subcutaneously (twice a week) with EC0565 (500nmol/kg) and everolimus (500 nmol/kg) on days 10, 13, 17, and 20. Theanimals in the healthy and arthritis control groups were left untreated.The arthritis scores (see FIG. 39) and animal body weights (see FIG. 40)were recorded three times a week. At the completion of study (day 24),the rats were euthanized by CO₂ asphyxiation and processed for paw andspleen weights (see FIGS. 41 and 42, respectively). Without being boundbe theory, it is believed that, given its low water solubility, thebioavailability of everolimus after subcutaneous administration waslower than that of EC0565. In this study, EC0565 was shown to be asactive or more active as everolimus against adjuvant-induced arthritis.

Example Radiographic Analysis Confirmed Less Tissue/Bone Damage inEC0565-Treated Arthritic Paws

AIA rats were treated subcutaneously (twice a week) with EC0565 (500nmol/kg), everolimus (500 nmol/kg), and methotrexate (190 nmol/kg) ondays 10, 13, 17, and 20. The animals in the healthy and arthritiscontrol groups were left untreated. At the completion of study (day 24),the rats were euthanized by CO₂ asphyxiation and the hind paws werefixed in 10% PBS-buffered formalin and subjected to radiographicanalysis. All radiographs were evaluated by a board-certifiedradiologist without knowledge of the assignment of treatment groups. Thefollowing radiographic changes were graded numerically according toseverity: increased soft tissue volume (0-4), narrowing or widening ofjoint spaces (0-5), subchondral erosion (0-3), periosteal reaction(0-4), osteolysis (0-4), subluxation (0-3) and degenerative jointchanges (0-3). The maximum possible score per foot was 26. In thisstudy, EC0565-treated rats showed minimal tissue and bone damage intheir hind paws when compared to arthritis control, everolimus-treated,and methotrexate-treated animals (see FIG. 43).

Example Anti-Arthritis Activity of EC0565 could be Partially Blocked byA Folate Competitor

The AIA rats were treated subcutaneously with EC0565 (500 nmol/kg) inthe absence (c) or presence (d) of 500-fold excess of EC0923 (250μmol/kg) on days 10, 13, and 17. The animals in the healthy (b) andarthritis control (a) groups were left untreated. The arthritis scores(see FIG. 44A) and animal body weights (see FIG. 44B) were recordedthree times a week. At the completion of study (day 24), the rats wereeuthanized by CO₂ asphyxiation and processed for paw (see FIG. 44C) andspleen (see FIG. 44D) weights. In this study, the anti-arthritisactivity of EC0565 was partially blocked by EC0923, a folate competitorwith similar affinity as free folic acid.

Example mTOR Knockdown

Western Blot Analysis. The data shown in FIG. 45 indicate that EC0565(folate-sugar-everolimus) can cause a dose-dependent, and specificknockdown of the downstream targets of mTOR (intracellular target foreverolimus). Without being bound by theory, in it believed that folatedelivers everolimus inside the cell where everolimus inhibits mTOR,which is the mammalian target of rapamycin and a ser/thr kinase.Inhibition of mTOR's downstream targets (P70 S6-kinase and Ribosomal S6)results, as shown on the Western blot.

Example Comparison of EC0565 with Oral Everolimus and SubcutaneousEtanercept in AIA Rats

Methotrexate (MTX) and etanercept (Enbrel®) are part of the currentstandard of care for RA. Treatment with EC0565 was compared to treatmentwith its base drug everolimus, MTX, and etanercept in rats with adjuvantarthritis using clinically relevant dosing routes. AIA rats were treated3 times a week with subcutaneous EC0565 (500 nmo/kg) and oral everolimus(500 nmol/kg) on days 10, 12, 14, 17, 19, and 21 post arthritisinduction. Biweekly oral MTX (250 nmol/kg) was given on days 10, 13, 17,and 20. Etanercept was given subcutaneously at 10 mg/kg once every 3days starting on day 10. At the completion of each study (day 24), ratswere euthanized by CO₂ asphyxiation and processed for paw weight (cut atthe hairline) and spleen weight. The removed hind paws wereimmersion-fixed in 10% buffered formalin and subjected to radiographicand histopathological analyses.

As shown in FIGS. 46A-46D, subcutaneously administered EC0565 (500nmol/kg/dose, 6 doses in 2 weeks) was more efficacious than oraleverolimus on an equimolar basis in the following clinical parametersassessed: arthritis score, percent change in body weight, and pawweight. EC0565 and everolimus-treated arthritic animals hadsignificantly decreased spleen weight compared to arthritic controls.The difference between treatment with EC0565 and treatment witherverolimus on spleen weight was not statistically different. EC0565 wasfound to be more effective than oral MTX and subcutaneous etanercept inall parameters assessed except for the effect on spleen weight.

Radiographic analysis of hind paws FIG. 47 revealed that EC0565-treatedrats had minimal tissue and bone damage in their hind paws when comparedto arthritis control, oral everolimus-treated, etanercept-treated, andoral MTX treated animals.

Histological grading of arthritis compared to the untreated controlsshowed oral everolimus-treated animals had significant reductions(44-61%) in all scored parameters (i.e. ankle inflammation, boneresorption, pannus formation, and cartilage damage) (FIG. 48A). Therewas a 52% significant decrease in the summed histological score (FIG.48B). Dorsal to ventral paw thickness was significantly decreased by 44%(FIG. 48C). While etanercept was not as effective as oral MTX, animalstreated with etanercept also had significant reductions in inflammation(42%) and bone resorption (55%), which contributed to a significant 43%decrease in the summed score (FIG. 48B) The dorsal to ventral pawthicknesses in both MTX and etanercept-treated rats were significantlydecreased by 63% and 40%, respectively (FIG. 48C). Animals treated withEC0565 had significant 88-100% decreases in all scored parameters (FIG.48A), with an overall 94% significant decrease in summed scores (FIG.48B). Dorsal to ventral paw thickness was significantly decreased by 94%(FIG. 48C). Overall, EC0565 consistently outperformed everolimus, MTX,and etanercept in all histological parameters assessed with furtherdecreases in the summed scores and dorsal to ventral paw thicknesses.The representative photomicrographs (16×) of the ankle closest to themean summed score for each group are shown in FIG. 49.

Example 2 EC0565 Anti-Arthritic Activity is Dose and Schedule-Dependent

To further investigate dose-response relationship andschedule-dependency, EC0565 was administered to rats with developing AIA(day 10) at 100, 500, and 1000 nmol/kg/dose (biweekly) or 1000nmol/kg/dose (once weekly). At completion of the study (day 24), ratswere euthanized by CO₂ asphyxiation and processed for paw weight (cut atthe hairline) and spleen weight. The differences in total paw weightbetween arthritic rats after treatment and that of healthy rats wereused as a measure of the extent of paw edema (FIG. 50A). Similarly, thedifferences in spleen weight between arthritic rats after treatment andthat of healthy rats were used as a measure of the extent ofsplenomegaly (FIG. 50B). As shown in FIG. 50A, EC0565 dosed for 2 weeksdisplayed a linear dose response in inhibiting paw edema from 100 to1000 nmol/kg/dose with an R-squared value of 0.862. As shown in FIG.50B, biweekly EC0565 treatment displayed a linear dose response ininhibiting splenomegaly from 0 to 500 nmol/kg/dose with an R-squaredvalue of 0.909 and no statistical difference between 500 and 1000nmol/kg was seen. When dosed once weekly for 2 weeks, EC0565 at 1000nmol/kg/dose remained highly effective, but this schedule did notcontrol the fast progressing AIA to the same degree as the biweeklyregimen at 500-1000 nmol/kg/dose (FIGS. 50A, B). Overall, EC0565 wasfound very effective against AIA and capable of controlling local(joints) and systemic (spleen) inflammation and halting the progressionof arthritis.

Example 3 Collagen-Induced Arthritis (CIA) Model

Collagen-induced arthritis (CIA) was induced in female Lewis rats onfolate-deficient diet (Harlan Teklad, Indianapolis, Ind.). On Day 0,rats were immunized with 500 μg of bovine collagen Type II (Chondrex,Redmond, Wash.) formulated with Freund's complete adjuvant. A boosterimmunization was given on day 7 with 250 lag of the bovine collagenformulated with Freund's incomplete adjuvant. Arthritis disease wasassessed by a qualitative clinical score system described by themanufacturer (Chondrex, Redmond, Wash.): 0=normal, 1=Mild, but definiteredness and swelling of the ankle or wrist, or apparent redness andswelling limited to individual digits, regardless of the number ofaffected digits, 2=Moderate redness and swelling of ankle of wrist,3=Severe redness and swelling of the entire paw including digits, and4=Maximally inflamed limb with involvement of multiple joints. On Day 10post first immunization, rats were distributed evenly (according to thearthritis score) across the control and treatment groups. The CIA ratswere given subcutaneous doses of EC0565 and everolimus at 1000 nmol/kg 3times a week. The animals in the arthritis control group were leftuntreated. The arthritis score and animal body weight were recordeddaily during weekdays. As shown in FIGS. 51A and 51B, EC0565 was alsoeffective in rats with collagen-induced arthritis.

Example 4 EC0565 Shows Higher Water Solubility and Bioavailability thanEverolimus in Rats

Everolimus, the base drug of EC0565 has poor water solubility (1-10 μM)and low and variable oral bioavailability (˜12% in rats, Journal ofPharmacokinetics and Pharmacodynamics, Vol. 34, No. 3, June 2007). Theselimitations render formulation of this drug difficult and contribute toa relatively narrow therapeutic index. In contrast, EC0565 displays aimproved water solubility at >1 mM in phosphate-buffered saline (pH7.4). The bioavailability of EC0565 after subcutaneous injection wasmeasured. Female Lewis rats with rounded tip jugular vein catheters(Harlan) were fed regular rodent diet. The treated animals were given asingle intravenous or subcutaneous dose of EC0565 at 2 μmol/kg. Forintravenous administration (FIG. 52A), whole blood samples (300 μl) werecollected from the animals at 1 min, 3 min, 7 min, 15 min, 30 min, 1h,2h, 4h, and 8h post injection. For subcutaneous administration (FIG.52B), whole blood samples (300 μl) were collected from the animals at 1min, 10 min, 30 min, 1h, 2h, 3h, 4h, 8h, and 12h post injection. Theblood samples were placed into anti-coagulant tubes containing 1.7 mg/mLof K3-EDTA and 0.35 mg/mL of N-maleoyl-beta-alanine (0.35 mg/mL). Plasmasamples were obtained by centrifugation for 3 min at ˜2,000 g and storedat −80° C. The amount of EC0565 and its released base drug (everolimus)were determined by HPLC using the EC0565 injection solution as thestandard. The results (based on the area under the curve) showed thatapproximately 18% and 17% of free drug exposure/release were detected inthe plasma after a single intravenous or subcutaneous dose of EC0565,respectively (FIGS. 52A, B). The Tmax for EC0565 and everolimus aftersubcutaneous injection were observed at ˜1 h (FIG. 52B). Based on thearea under the curve, the bioavailablity of EC0565 after subcutaneousadministration (compared to intravenous administration) was calculatedto be ˜128% (FIG. 52C).

Example 5 EC0565 Inhibits Proliferating Cell Nuclear Antigen in RAW264.7Cells

Proliferating Cell Nuclear Antigen (PCNA) is a cell-cycle regulatednuclear protein that is often used to evaluate cellular proliferativeactivity. To study anti-proliferative effect of EC0565, FR-positivemurine macrophage-like RAW264.7 cells (serum deprived for 36h forsynchonization) were given a 2 h treatment of EC0565 (1, 10, 100, and1000 nM) without or with 1000× excess of EC17 (as a folate competitor)followed by a 48-h chase in drug-free medium. For comparison, the cellswere also treated for 48 h with everolimus (1, 10, 100, and 1000 nM).All media contained 1% DMSO due to the low water solubility ofeverolimus. Western blot analysis was carried out on whole cell lysatesusing a monoclonal antibody specific for PCNA (PC10, Cell Signaling,Danvers, Mass.). After incubation with a peroxidase-conjugated secondaryantibody, the signals were visualized using SuperSignal West PicoChemiluminescent Substrate system (ThermoScientific, Waltham, Mass.)following the manufacturer's instructions. The images were acquiredusing a G:BOX Chemi HR 16 gel imaging system (Syngene, Frederick, Md.).As shown in FIG. 53A, both everolimus and EC0565 inhibited PCNAactivities in the synchronized RAW264.7 cells. The inhibitory activityof EC0565 at 1 nM was 100% blocked by the presence of excess EC17, whileEC17 alone was benign (FIGS. 53B-C). As the EC0565 concentration wasincreased from 10, 100, to 1000 nM, EC0565 showed both FR-specific andnon FR-specific anti-PCNA effects. This data indicates that EC0565reduces PCNA activity in RAW264.7 cells in a FR-dependent manner,especially at lower concentrations.

COMPOUND EXAMPLES

Example

(3,4), (5,6)-Bisacetonide-D-Gluconic Acid Methyl Ester. In a dry 250 mLround bottom flask, under argon 6-gluconolactone (4.14 g, 23.24 mmol)was suspended in acetone-methanol (50 mL). To this suspensiondimethoxypropane (17.15 mL, 139.44 mmol) followed by catalytic amount ofp-toulenesulfonic acid (200 mg) were added. This solution was stirred atroom temperature for 16 h. TLC (50% EtOAc in petroleum ether) showedthat all of the starting material had been consumed and product had beenformed. Acetone-methanol was removed under reduced pressure. The residueof the reaction was dissolved in EtOAc and washed with water. Theorganic layer was washed with brine, dried over Na₂SO₄, and concentratedto dryness. This material was then loaded onto a SiO₂ column andchromatographed (30% EtOAc in petroleum ether) to yield pure (3,4),(5,6)-bisacetonide-D-gluconic acid methyl ester (3.8 g, 56%) andregio-isomer (2,3), (5,6)-bisacetonide-D-gluconic acid methyl ester(0.71 g, 10%). ¹H NMR data was in accordance with the required products.C₁₃H₂₂O₇; MW 290.31; Exact Mass: 290.14.

Example

(3,4), (5,6)-Bisacetonide-D-Gluconic Amide. 20 g of the methyl ester wasdissolved in 100 mL methanol, cooled the high-pressure reaction vesselwith dry ice/acetone, charged with 100 mL liquid ammonia, warmed up toroom temperature and heated to 160° C./850 PSI for 2 hours. The reactionvessel was cooled to room temperature and released the pressure.Evaporation of the solvent gave brownish syrup, and minimum amount ofisopropyl alcohol was added to make the homogeneous solution withreflux. The solution was cooled to −20° C. and the resulting solid wasfiltered to give 8.3 g of solid. The mother liquid was evaporated, andto the resulting residue, ether was added and refluxed until homogeneoussolution was achieved. The solution was then cooled to −20° C. and theresulting solid was filtered to give 4.0 g product. The solid wascombined and recrystallized in isopropyl alcohol to give 11.2 g (59%) ofthe white amide product. C₁₂H₂₁NO₆; MW 275.30; Exact Mass: 275.14.

Example

(3,4), (5,6)-Bisacetonide-1-Deoxy-1-Amino-D-Glucitol. In a dry 100 mLround bottom flask, under argon, LiAlH₄ (450 mg, 11.86 momol)) wasdissolved in THF (10 mL) and cooled to 0° C. To this suspension (3,4),(5,6)-bisacetonide-D-gluconic amide (1.09 g, 3.96 mmol) in THF (30 mL)was added very slowly over 15 min. This mixture was refluxed for 5 h.TLC (10% MeOH in methylene chloride) showed that all of the startingmaterial had been consumed and product had been formed. The reactionmixture was cooled to room temperature, and then cooled to ice-bathtemperature, diluted with diethyl ether (40 mL), slowly added 0.5 mL ofwater, 0.5 mL of 15% aq. NaOH, and then added 1.5 mL of water. Thereaction mixture was warmed to room temperature and stirred for 30 min.MgSO₄ was added and stirred for additional 15 min and filtered. Theorganic layer was concentrated to dryness to yield (3,4),(5,6)-bisacetonide-1-deoxy-1-amino-D-glucitol. ¹H NMR data was inaccordance with the product. C₁₂H₂₃NO₅; MW 261.31; Exact Mass: 261.16.

Example

EC0475. O-Allyl protected Fmoc-Glu (2.17 g, 1 eq), PyBOP (2.88 g, 1 eq),and DIPEA (1.83 mL, 2 eq) were added to a solution of(3,4),(5,6)-bisacetonide-1-deoxy-1-amino-D-glucitol (1.4 g, 5.3 mmol) indry DMF (6 mL) and the mixture was stirred at RT under Ar for 2 h. Thesolution was diluted with EtOAc (50 mL), washed with brine (10 mL×3),organic layer separated, dried (MgSO₄), filtered and concentrated togive a residue, which was purified by a flash column (silica gel, 60%EtOAc/petro-ether) to afford 1.72 g (50%) allyl-protected EC0475 as asolid. Pd(Ph₃)₄ (300 mg, 0.1 eq) was added to a solution ofallyl-protected EC0475 (1.72 g, 2.81 mmol) in NMM/AcOH/CHCl₃ (2 mL/4mL/74 mL). The resulting yellow solution was stirred at RT under Ar for1 h, to which was added a second portion of Pd(Ph₃)₄ (300 mg, 0.1 eq).After stirring for an additional 1 h, the mixture was washed with 1 NHCl (50 mL×3) and brine (50 mL), organic layer separated, dried (MgSO4),filtered, and concentrated to give a yellow foamy solid, which wassubject to chromatography (silica gel, 1% MeOH/CHCl₃ followed by 3.5%MeOH/CHCl₃) to give 1.3 g (81%) EC0475 as a solid material. MW 612.67;Exact Mass: 612.27.

Example

Tetra-Saccharoglutamate-Bis-αGlu-Folate Spacer EC0491. EC0491 wassynthesized by SPPS in eight steps according to the general peptidesynthesis procedure described herein starting fromH-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPSreagents:

Reagents Mmol equivalent MW Amount H-Cys(4-methoxytrityl)- 0.1 0.167 g2-chlorotrityl-Resin (loading 0.56 mmol/g) EC0475 0.13 1.3 612.67 0.080g Fmoc-Glu(OtBu)-OH 0.2 2 425.5 0.085 g EC0475 0.13 1.3 612.67 0.080 gEC0475 0.13 1.3 612.67 0.080 g Fmoc-Glu(OtBu)-OH 0.2 2 425.5 0.085 gEC0475 0.13 1.3 612.67 0.080 g Fmoc-Glu-OtBu 0.2 2 425.5 0.085 gN¹⁰TFA-Pteroic 0.2 2 408 0.105 g Acid•TFA (dissolve in 10 ml DMSO) DIPEA0.4 4 129.25 0.070 mL (d = 0.742) PyBOP 0.2 2 520 0.104 gThe Coupling steps, Cleavage step, and Cleavage Reagent were identicalto those described above. HPLC Purification step: Column: Waters NovaPakC₁₈ 300×19 mm; Buffer A=10 mM ammonium acetate, pH 5; B=ACN; Method:100% A for 5 min then 0% B to 20% B in 20 minutes at 26 ml/min;yield˜100 mg, 51%. C₇₆H₁₁₈N₁₈O₄₁S; MW 1971.91; Exact Mass: 1970.74.

Example

EC0479 was synthesized by SPPS according to the general peptidesynthesis procedure described herein starting fromH-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPSreagents:

Reagents mmol equivalent MW AmountH-Cys(4-methoxytrityl)-2-chlorotrityl- 0.094  0.16 g Resin (loading 0.6mmol/g) EC0475 0.13 1.4 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19 2.0 425.470.080 g EC0475 0.13 1.4 612.67 0.082 g Fmoc-Arg(Pbf)-OH 0.19 2.0 648.77 0.12 g EC0475 0.13 1.4 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19 2.0 425.470.080 g EC0475 0.13 1.4 612.67 0.082 g Fmoc-Glu-OtBu 0.19 2.0 425.470.080 g N¹⁰TFA-Pteroic Acid 0.16 1.7 408.29 0.066 g (dissolve in 10 mlDMSO) DIPEA 2.0 eq of AA 41 μL or 49 μL PyBOP 1.0 eq of AA 122 mg or 147mg

Coupling steps. In a peptide synthesis vessel add the resin, add theamino acid solution, DIPEA, and PyBOP. Bubble argon for 1 hr. and wash3× with DMF and IPA. Use 20% piperidine in DMF for Fmoc deprotection, 3×(10 min), before each amino acid coupling. Continue to complete all 9coupling steps. At the end treat the resin with 2% hydrazine in DMF 3×(5 min) to cleave TFA protecting group on Pteroic acid, wash the resinwith DMF (3×), IPA (3×), MeOH (3×), and bubble the resin with argon for30 min.

Cleavage step. Reagent: 92.5% TFA, 2.5% H₂O, 2.5% triisopropylsilane,2.5% ethanedithiol. Treat the resin with cleavage reagent for 15 minwith argon bubbling, drain, wash the resin once with cleavage reagent,and combine the solution. Rotavap until 5 ml remains and precipitate indiethyl ether (35 mL). Centrifuge, wash with diethyl ether, and dry. Thecrude solid was purified by HPLC.

HPLC Purification step. Column: Waters Atlantis Prep T3 10 m OBD 19×250mm; Solvent A: 10 mM ammonium acetate, pH 5; Solvent B: ACN; Method: 5min 0% B to 20 min 20% B 26 mL/min. Fractions containing the product wascollected and freeze-dried to give ˜70 mg EC0479 (35% yield). ¹H NMR andLC/MS were consistent with the product. MW 2128.10; Exact Mass: 2126.84.

EC0488. This compound was prepared by SPPS according to the generalpeptide synthesis procedure described herein starting fromH-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPSreagents:

Reagents mmol equivalent MW amount H-Cys(4-methoxytrityl)- 0.10  0.17 g2-chlorotrityl-Resin (loading 0.6 mmol/g) EC0475 0.13 1.3 612.67 0.082 gFmoc-Glu(OtBu)-OH 0.19 1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 gFmoc-Glu(OtBu)-OH 0.19 1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 gFmoc-Glu-OtBu 0.19 1.9 425.47 0.080 g N¹⁰TFA-Pteroic Acid 0.16 1.6408.29 0.066 g (dissolve in 10 ml DMSO) DIPEA 2.0 eq of AA PyBOP 1.0 eqof AA

Coupling steps. In a peptide synthesis vessel add the resin, add theamino acid solution, DIPEA, and PyBOP. Bubble argon for 1 hr. and wash3× with DMF and IPA. Use 20% piperidine in DMF for Fmoc deprotection, 3×(10 min), before each amino acid coupling. Continue to complete all 9coupling steps. At the end treat the resin with 2% hydrazine in DMF 3×(5 min) to cleave TFA protecting group on Pteroic acid, wash the resinwith DMF (3×), IPA (3×), MeOH (3×), and bubble the resin with argon for30 min.

Cleavage step. Reagent: 92.5% TFA, 2.5% H₂O, 2.5% triisopropylsilane,2.5% ethanedithiol. Treat the resin with cleavage reagent 3× (10 min, 5min, 5 min) with argon bubbling, drain, wash the resin once withcleavage reagent, and combine the solution. Rotavap until 5 ml remainsand precipitate in diethyl ether (35 mL). Centrifuge, wash with diethylether, and dry. About half of the crude solid (˜100 mg) was purified byHPLC.

HPLC Purification step. Column: Waters Xterra Prep MS C18 10 μm 19×250mm; Solvent A: 10 mM ammonium acetate, pH 5; Solvent B: ACN; Method: 5min 0% B to 25 min 20% B 26 mL/min. Fractions containing the product wascollected and freeze-dried to give 43 mg EC0488 (51% yield). ¹H NMR andLC/MS (exact mass 1678.62) were consistent with the product. MW 1679.63;Exact Mass: 1678.62.

EC0536 Conjugate Intermediate

EC0632 Conjugate intermediate. C52H72N14O28S, MW 1373.27, Exact Mass:1372.44, prepared from the corresponding tert-butyl protectedcarboxylates.

EC0669 Conjugate intermediate. C49H71N13O24S, MW 1258.23, Exact Mass:1257.45

Example

Synthesis of Coupling Reagent EC0311. DIPEA (0.60 mL) was added to asuspension of HOBt-OCO₂—(CH₂)₂-SS-2-pyridine HCl (685 mg, 91%) inanhydrous DCM (5.0 mL) at 0° C., stirred under argon for 2 minutes, andto which was added anhydrous hydrazine (0.10 mL). The reaction mixturewas stirred under argon at 0° C. for 10 minutes and room temperature foran additional 30 minutes, filtered, and the filtrate was purified byflash chromatography (silica gel, 2% MeOH in DCM) to afford EC0311 as aclear thick oil (371 mg), solidified upon standing.

EC0593 Multidrug intermediate for two drugs. C68H103N17O35S2, MW1782.77, Exact Mass: 1781.62

EC0613 Multidrug intermediate for three drugs. C90H140N22O47S4, MW2410.45, Exact Mass: 2408.81

EC0542 Optionally selective multidrug intermediate for two drugs.C85H118N18O36S2, C, 50.24; H, 5.85; N, 12.41; 0, 28.34; S, 3.16, MW2032.08, Exact Mass: 2030.74

EC0559 Optionally selective multidrug intermediate for two drugs.C90H121N19O36S3, MW 2141.22, Exact Mass: 2139.74

EC0682 Optionally selective multidrug intermediate for two drugs.C95H132N20O42S2, MW 2290.30, Exact Mass: 2288.82

EC0646 Conjugate of Aminopterin and intermediate for multidrugconjugate. C106H140N26O41S3, MW 2530.59, Exact Mass: 2528.88

Example

EC0746 Conjugate of aminopterin. C₈₇H₁₂₂N₂₆O₄₀S₂; C, 46.73; H, 5.50; N,16.29; 0, 28.62; S, 2.87; MW 2236.180, Exact Mass: 2234.775.

Reaction of mixed carbonate 101 with t-butyl-carbazate in the presenceof diisopropylethylamine (DIPEA) gave the correspondingt-butyl-carbazate 102. Trifluoroacetic acid (TFA) mediated Bocdeprotection of 102 in the presence of triisopropylsilane (TIPS)resulted in pyridyldisulfanylethyl carbazate 103 as a TFA salt. Couplingof carbazate 103 with protected glutamic acid 104, usingbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBop) and DIPEA, yielded glutamic acid derivative 105.4-dimethylaminopyridine (DMAP) mediated Fmoc deprotection of 105followed by in situ coupling with commercially available4-[(2-amino-4-imino-3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoicacid 106 using PyBop and hydroxybenzotriazol (HOBt) resulted inprotected aminopterin hydrazide 107. Treatment of 107 with TFA removedthe t-butyl moiety to yield pyridinedisulfanyl-activated aminopterinhydrazide 108. ¹H NMR (DMSO-d₆ & D₂O) δ 8.82 (s, 1H), 8.44 (d, J=4.7 Hz,1H), 7.80 (m, 2H), 7.71 (d, J=8.8 Hz, 2H), 7.24 (t, J=4.6 Hz, 1H), 6.74(d, J=8.8 Hz, 2H), 4.60 (s, 2H), 4.32 (dd, J=5.0 Hz, 1H), 4.20 (t, J=6.0Hz, 2H), 3.07 (t, J=6.0 Hz, 2H), 2.22 (t, J=7.8 Hz, 2H), 2.15-1.94 (m,2H). ESI-MS: (M+H)⁺=Calculated 668.2; found 668.2. Treatment of asuspension of EC0488 in phosphate buffer under argon with NaHCO₃resulted in a clear yellow solution. A dimethylsulfoxide (DMSO) solutionof 108 was added to this mixture at once under vigorous stirring toyield EC0746. ¹H NMR (DMSO-d₆ & D₂O) δ 8.67 (s, 1H) 8.60 (s, 1H), 7.62(d, J=9 Hz, 2H), 7.59 (d, J=9 Hz, 2H), 6.71 (d, J=8.7 Hz, 2H), 6.62 (d,J=8.1 Hz, 2H), 4.47 (m, 4H), 4.26-4.04 (m, 10H), 3.70-3.30 (m, 22H),3.30-3.10 (m, 6H), 3.10-2.76 (m, 9H), 2.40-2.04 (m, 15H), 2.04-1.60 (m,4H). ESI-MS: [(M+2H)²+]/2=Calculated 1119.09; found 1119.10.

EC0808 is an isomer of EC0746 having the opposite configuration at thestereogenic carbon indicated with the arrow.

EC0932, everolimus-aminopterin hydrazide conjugate C149H218N30O57S4; C,51.58; H, 6.33; N, 12.11; 0, 26.28; S, 3.70; MW 3469.752; Exact Mass:3467.396.

EC0828 everolimus-aminopterin hydrazide conjugate. C149H217N29O58S4; C,51.56; H, 6.30; N, 11.70; 0, 26.74; S, 3.70; MW: 3470.737; Exact Mass:3468.381.

Example

Everolimus (2′-pyridyldisulfanyl)ethyl carbonate (EC0564). In a 10 mLround bottom flask, under argon atmosphere, everolimus (130 mg, 0.136mmol) was dissolved in 2.0 mL of CH₂Cl₂.2-[Benzotriazole-1-yl-(oxycarbonyloxy)-ethyldisulfanyl]-pyridine (104.4mg, 0.271 mmol) followed by DMAP (49.85 mg, 0.41 mmol) were added. Thereaction mixture was stirred for 30 min. Progress of the reaction wasmonitored by analytical HPLC (0.1% TFA in water, pH=2.0 andacetonitrile). The reaction mixture was diluted with CH₂Cl₂ and washedwith sat. NH₄Cl. The organic layer was dried over Na₂SO₄ andconcentrated to yield everolimus (2′-pyridyldisulfanyl)ethyl carbonate,EC0564.

Example

Everolimus-EC0488 conjugate (EC0565). In a 25 mL round bottom flask,folate linker (EC0488, 104 mg, 0.06 mmol) was dissolved in 2.0 mL ofDMSO, and 0.13 mL of DIPEA (20 equiv) were added. The everolimuscarbonate derivative (EC0564, 38 mg, 1.0 eq) in 1.0 mL of DMSO was addedquickly to the above solution. The resulting clear solution was stirredunder argon. Progress of the reaction was monitored by analytical HPLC(20 mM NH₄OAc buffer, pH=5.0 and acetonitrile). After 20 min, reactionmixture was injected on a prep-HPLC. HPLC purificationconditions—column: Waters X-Bridge Prep MS C₁₈ 10 m 19×100 mm; solventA: 20 mM ammonium acetate, pH 5; solvent B: acetonitrile; method: 5 min10% B to 25 min 80% B 25 mL/min. Fractions containing EC0565 werecollected and freeze-dried to afford 68 mg (50% yield, over 2 steps fromeverolimus) of fluffy yellow solid. C₁₂₁H₁₈₃N₁₇O₅₀S₂; C, 53.04; H, 6.73;N, 8.69; 0, 29.20; S, 2.34; MW 2739.96; Exact Mass: 2738.17.

EC0606 Conjugate of Everolimus and intermediate for multidrug conjugate.C141H203N19O52S3, C, 54.76; H, 6.62; N, 8.61; 0, 26.90; S, 3.11, MW3092.42, Exact Mass: 3090.30

EC0634 Intermediate for optional non-targeted delivery. C63H95N9O30S2,MW 1522.60, Exact Mass: 1521.56

EC0586 Intermediate for optional non-target delivery. C48H83N9O30S, MW1298.28, Exact Mass: 1297.50

EC0539 Conjugate of Lysine Analog of Aminopterin

EC0544 Conjugate of cysteine analog of aminopterin. C83H116N24O37S2, C,47.33; H, 5.55; N, 15.96; 0, 28.11; S, 3.05, MW 2106.08, Exact Mass:2104.74

EC0551 Conjugate of aminopterin. C86H120N24O39S2, C, 47.42; H, 5.55; N,15.43; 0, 28.65; S, 2.94, MW 2178.14, Exact Mass: 2176.76

EC0647 Bis aminopterin conjugate. C110H147N33O45S4, MW, 2779.80, ExactMass: 2777.9112, m/z: 2778.91 (100.0%), 2777.91 (74.4%), 2779.92 (62.2%)

1-2. (canceled)
 3. A compound of the formulaBLA or a pharmaceutically acceptable salt thereof; wherein B is avitamin receptor binding ligand; L is a linker that comprises one ormore hydrophilic spacer linkers selected from the group consisting of

wherein R is selected from the group consisting of H, alkyl, cycloalkyl,and arylalkyl; m is an integer from 1 to about 3; n is an integer from 1to about 6; p is an integer from 1 to about 5; and (*) indicates thepoint of attachment to the rest of the compound and a fragment of theformula

wherein each R^(a) and R^(b) is independently hydrogen or alkyl; orR^(a) and R^(b) are taken together with the attached carbon atom to forma carbocyclic ring; n¹ is an integer selected from 1 to 4; and (**)indicates points of attachment for other parts of the conjugate; and Ais an adrenocorticoid or a corticosteroid.
 4. The compound of claim 3 ora pharmaceutically acceptable salt thereof, wherein A is anadrenocorticoid.
 5. The compound of claim 3 or a pharmaceuticallyacceptable salt thereof, wherein A is a corticosteroid.
 6. The compoundof claim 3 or a pharmaceutically acceptable salt thereof, wherein thelinker comprises three hydrophilic spacer linkers.
 7. The compound ofclaim 3 or a pharmaceutically acceptable salt thereof, wherein the oneor more hydrophilic spacer linkers each comprise five hydroxyl groups.8. The compound of claim 3 or a pharmaceutically acceptable saltthereof, wherein the linker further comprises one or more amino acidsselected from the group consisting of the naturally occurring aminoacids and stereoisomers thereof.
 9. The compound of claim 8 or apharmaceutically acceptable salt thereof, wherein the linker furthercomprises one or more amino acids selected from the group consisting ofasparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine,arginine, serine, ornithine, threonine, and stereoisomers thereof. 10.The compound of claim 3 or a pharmaceutically acceptable salt thereof,wherein the linker further comprises a releasable linker.
 11. Thecompound of claim 10 or a pharmaceutically acceptable salt thereof,wherein the linker further comprises a disulfide group.
 12. The compoundof claim 3 or a pharmaceutically acceptable salt thereof, wherein thepurity of the compound is at least 98%.
 13. The compound of claim 3 or apharmaceutically acceptable salt thereof, wherein the vitamin receptorbinding ligand is a folate.
 14. The compound of claim 4 or apharmaceutically acceptable salt thereof, wherein the vitamin receptorbinding ligand is a folate.
 15. The compound of claim 5 or apharmaceutically acceptable salt thereof, wherein the vitamin receptorbinding ligand is a folate.
 16. The compound of claim 13 or apharmaceutically acceptable salt thereof, wherein the vitamin receptorbinding ligand is of the formula

wherein * indicates the point of attachment to the linker.
 17. Thecompound of claim 14 or a pharmaceutically acceptable salt thereof,wherein the vitamin receptor binding ligand is of the formula

wherein * indicates the point of attachment to the linker.
 18. Thecompound of claim 15 or a pharmaceutically acceptable salt thereof,wherein the vitamin receptor binding ligand is of the formula

wherein * indicates the point of attachment to the linker.
 19. Apharmaceutical composition comprising a compound of the formulaBLA or a pharmaceutically acceptable salt thereof; wherein B is avitamin receptor binding ligand; L is a linker that comprises one ormore hydrophilic spacer linkers selected from the group consisting of

wherein R is selected from the group consisting of H, alkyl, cycloalkyl,and arylalkyl; m is an integer from 1 to about 3; n is an integer from 1to about 6; p is an integer from 1 to about 5; and (*) indicates thepoint of attachment to the rest of the compound and a fragment of theformula

wherein each R^(a) and R^(b) is independently hydrogen or alkyl; orR^(a) and R^(b) are taken together with the attached carbon atom to forma carbocyclic ring; n¹ is an integer selected from 1 to 4; and (**)indicates points of attachment for other parts of the conjugate; and Ais an adrenocorticoid or a corticosteroid.
 20. The pharmaceuticalcomposition of claim 19, wherein, in the compound, or thepharmaceutically acceptable salt thereof, the vitamin receptor bindingligand is a folate and A is an adrenocorticoid.
 21. The pharmaceuticalcomposition of claim 19, wherein, in the compound, or thepharmaceutically acceptable salt thereof, the vitamin receptor bindingligand is a folate and A is a corticosteroid.
 22. The pharmaceuticalcomposition of claim 19, further comprising one or more carriers,diluents, or excipients, or a combination thereof.